Circular permuteins of flt3 ligand

ABSTRACT

Disclosed are novel flt-3 receptor agonist proteins, DNAs which encode the flt-3 receptor agonist proteins, methods of making the flt-3 receptor agonist proteins and methods of using the flt-3 receptor agonist proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. patentapplication Ser. No. 08/955,090, filed Oct. 21, 1997, pending; whichclaims priority under Title 35, United States Code, § 119(e) (1) of U.S.Prov. Pat. App. Ser. No. 60/030,094, filed Oct. 25, 1996.

FIELD OF THE INVENTION

[0002] The present invention relates to human flt3 receptor agonists.These flt3 receptor agonists retain one or more activities of nativeflt3 ligand and may also show improved hematopoietic cell-stimulatingactivity and/or an improved activity profile which may include reductionof undesirable biological activities associated with native flt3 ligandand/or have improved physical properties which may include increasedsolubility, stability and refold efficiency.

REFERENCE TO A “SEQUENTIAL LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISC

[0003] This application includes a computer program listing appendix,pursuant to 37 CFR 1.96, contained on a compact disc, which isincorporated fully into this application by this reference.

[0004] The compact disc is labeled as follows:

[0005] Inventors: Charles A. McWherter, John P. McKearn, Philip R.Streeter, Nancy I. Minster, Yiquing Feng, Nicholas R. Staten, Susan L.Woulfe, & John C. Minnerly

[0006] Title: Circular Permuteins of FLT3 Ligand

[0007] Attorney docket number: 126181-1059

[0008] Creation date of the compact disc: Aug. 20, 2003

[0009] The compact disc contains the following files in ASCII fileformat:

[0010] File Name File size Creation Date

[0011] Seq List 2993-3 US.txt 118 kb Aug. 20, 2003

BACKGROUND OF THE INVENTION

[0012] Colony stimulating factors which stimulate the differentiationand/or proliferation of bone marrow cells have generated much interestbecause of their therapeutic potential for restoring depressed levels ofhematopoietic stem cell-derived cells. Colony stimulating factors inboth human and murine systems have been identified and distinguishedaccording to their activities. For example, granulocyte-CSF (G-CSF) andmacrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilicgranulocyte and macrophage colonies, respectively while GM-CSF andinterleukin-3 (IL-3) have broader activities and stimulate the formationof both macrophage, neutrophilic and eosinophilic granulocyte colonies.Certain factors such as flt3 ligand are able to predominately affectstem cells.

[0013] Tyrosine kinase receptors are growth factor receptors thatregulate the proliferation and differentiation of a number of cell.Certain tyrosine kinase receptors function within the hematopoieticsystem. Flt3 ligand (Rosnet et al., Oncogene, 6:1641-1650, 1991) andflk-2 (Matthews et al., Cell, 65:1143-1152, 1991) are forms of atyrosine kinase receptor that is related to c-fms and c-kit receptors.The flk-2 and flt3 receptors are similar in amino acid sequence and varyat two amino acid residues in the extracellular domain and diverge in a31 amino acid segment located near the C-terminus.

[0014] flt3 ligand is a hematopoietic growth factor which has theproperty of being able to regulate the growth and differentiation ofhematopoietic progenitor and stem cells. Because of its ability tosupport the growth and proliferation of progenitor cells, flt3 receptoragonists have potential for therapeutic use in treating hematopoieticdisorders such as aplastic anemia and myelodysplastic syndromes.Additionally, flt3 receptor agonists will be useful in restoringhematopoietic cells to normal amounts in those cases where the number ofcells has been reduced due to diseases or to therapeutic treatments suchas radiation and chemotherapy.

[0015] WO 94/28391 discloses the native flt3 ligand protein sequence anda cDNA sequence encoding the flt3 ligand, methods of expressing flt3ligand in a host cell transfected with the cDNA and methods of treatingpatients with a hematopoietic disorder using flt3 ligand.

[0016] U.S. Pat. No. 5,554,512 is directed to human flt3 ligand as anisolated protein, DNA encoding the flt3 ligand, host cells transfectedwith cDNAs encoding flt3 ligand and methods for treating patients withflt3 ligand.

[0017] WO 94/26891 provides mammalian flt3 ligands, including an isolatethat has an insertion of 29 amino acids, and fragments there of.

Rearrangement of Protein Sequences

[0018] In evolution, rearrangements of DNA sequences serve an importantrole in generating a diversity of protein structure and function. Geneduplication and exon shuffling provide an important mechanism to rapidlygenerate diversity and thereby provide organisms with a competitiveadvantage, especially since the basal mutation rate is low (Doolittle,Protein Science 1:191-200, 1992).

[0019] The development of recombinant DNA methods has made it possibleto study the effects of sequence transposition on protein folding,structure and function. The approach used in creating new sequencesresembles that of naturally occurring pairs of proteins that are relatedby linear reorganization of their amino acid sequences (Cunningham, etal., Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222, 1979; Teather & Erfle,J. Bacteriol. 172: 3837-3841, 1990; Schimming et al., Eur. J. Biochem.204: 13-9, 1992; Yamiuchi and Minamikawa, FEBS Lett. 260:127-130, 1991:MacGregor et al., FEBS Lett. 378:263-266, 1996). The first in vitroapplication of this type of rearrangement to proteins was described byGoldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). A newN-terminus is selected at an internal site (breakpoint) of the originalsequence, the new sequence having the same order of amino acids as theoriginal from the breakpoint until it reaches an amino acid that is ator near the original C-terminus. At this point the new sequence isjoined, either directly or through an additional portion of sequence(linker), to an amino acid that is at or near the original N-terminus,and the new sequence continues with the same sequence as the originaluntil it reaches a point that is at or near the amino acid that wasN-terminal to the breakpoint site of the original sequence, this residueforming the new C-terminus of the chain.

[0020] This approach has been applied to proteins which range in sizefrom 58 to 462 amino acids (Goldenberg & Creighton, J. Mol. Biol.165:407-413, 1983; Li & Coffino, Mol. Cell. Biol. 13:2377-2383, 1993).The proteins examined have represented a broad range of structuralclasses, including proteins that contain predominantly α-helix(interleukin-4; Kreitman et al., Cytokine 7:311-318, 1995), β-sheet(interleukin-1; Horlick et al., Protein Eng. 5:427-431, 1992), ormixtures of the two (yeast phosphoribosyl anthranilate isomerase; Lugeret al., Science 243:206-210, 1989). Broad categories of protein functionare represented in these sequence reorganization studies: Enzymes T4lysozyme Zhang et al., Biochemistry 32: 12311-12318 (1993); Zhang etal., Nature Struct. Biol. 1: 434-438 (1995) dihydrofolate Buchwalder etal., Biochemistry reductase 31: 1621-1630 (1994); Protasova et al.,Prot. Eng. 7: 1373-1377 (1995) ribonuclease T1 Mullins et al., J. Am.Chem. Soc. 116: 5529-5533 (1994); Garrett et al., Protein Science 5:204-211 (1996) Bacillus β-glucanse Hahn et al., Proc. Natl. Acad. Sci.U.S.A. 91: 10417-10421 (1994) aspartate Yang & Schachman, Proc. Natl.Acad. transcarbamoylase Sci. U.S.A. 90: 11980-11984 (1993)phosphoribosyl Luger et al., Science 243: 206-210 anthranilate (1989);Luger et al., Prot. Eng. isomerase 3: 249-258 (1990) pepsin/pepsinogenLin et al., Protein Science 4: 159-166 (1995) glyceraldehyde-3- Vignaiset al., Protein Science phosphate dehydrogenase 4: 994-1000 (1995)ornithine Li & Coffino, Mol. Cell. Biol. decarboxylase 13: 2377-2383(1993) yeast Ritco-Vonsovici et al., Biochemistry phosphoglycerate 34:16543-16551 (1995) dehydrogenase Enzyme Inhibitor basic pancreaticGoldenberg & Creighton, J. Mol. trypsin inhibitor Biol. 165: 407-413(1983) Cytokines interleukin-1β Horlick et al., Protein Eng. 5: 427-431(1992) interleukin-4 Kreitman et al., Cytokine 7: 311-318 (1995)Tyrosine Kinase Recognition Domain α-spectrin SH3 Viguera, et al., J.domain Mol. Biol. 247: 670-681 (1995) Transmembrane Protein omp AKoebnik & Krämer, J. Mol. Biol. 250: 617-626 (1995) Chimeric Proteininterleukin-4- Kreitman et al., Proc. Natl. Acad. Pseudomonas Sci.U.S.A. 91: 6889-6893 (1994) exotoxin fusion molecule

[0021] The results of these studies have been highly variable. In manycases substantially lower activity, solubility or thermodynamicstability were observed (E. coli dihydrofolate reductase, aspartatetranscarbamoylase, phosphoribosyl anthranilate isomerase,glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, ompA, yeast phosphoglycerate dehydrogenase). In other cases, the sequencerearranged protein appeared to have many nearly identical properties asits natural counterpart (basic pancreatic trypsin inhibitor, T4lysozyme, ribonuclease T1, Bacillus β-glucanase, interleukin-1⊕,α-spectrin SH3 domain, pepsinogen, interleukin-4). In exceptional cases,an unexpected improvement over some properties of the natural sequencewas observed, e.g., the solubility and refolding rate for rearrangedα-spectrin SH3 domain sequences, and the receptor affinity andanti-tumor activity of transposed interleukin-4-Pseudomonas exotoxinfusion molecule (Kreitman et al., Proc. Natl. Acad. Sci. U.S.A.91:6889-6893, 1994; Kreitman et al., Cancer Res. 55:3357-3363, 1995).

[0022] The primary motivation for these types of studies has been tostudy the role of short-range and long-range interactions in proteinfolding and stability. Sequence rearrangements of this type convert asubset of interactions that are long-range in the original sequence intoshort-range interactions in the new sequence, and vice versa. The factthat many of these sequence rearrangements are able to attain aconformation with at least some activity is persuasive evidence thatprotein folding occurs by multiple folding pathways (Viguera, et al., J.Mol. Biol. 247:670-681, 1995). In the case of the SH3 domain ofα-spectrin, choosing new termini at locations that corresponded toβ-hairpin turns resulted in proteins with slightly less stability, butwhich were nevertheless able to fold.

[0023] The positions of the internal breakpoints used in the studiescited here are found exclusively on the surface of proteins, and aredistributed throughout the linear sequence without any obvious biastowards the ends or the middle (the variation in the relative distancefrom the original N-terminus to the breakpoint is ca. 10 to 80% of thetotal sequence length). The linkers connecting the original N- andC-termini in these studies have ranged from 0 to 9 residues. In one case(Yang & Schachman, Proc. Natl. Acad. Sci. U.S.A. 90:11980-11984, 1993),a portion of sequence has been deleted from the original C-terminalsegment, and the connection made from the truncated C-terminus to theoriginal N-terminus. Flexible hydrophilic residues such as Gly and Serare frequently used in the linkers. Viguera, et al. (J. Mol. Biol.247:670-681, 1995) compared joining the original N- and C-termini with3- or 4-residue linkers; the 3-residue linker was less thermodynamicallystable. Protasova et al. (Protein Eng. 7:1373-1377, 1994) used 3- or5-residue linkers in connecting the original N-termini of E. colidihydrofolate reductase; only the 3-residue linker produced protein ingood yield.

SUMMARY OF THE INVENTION

[0024] The modified human flt3 receptor agonists of the presentinvention can be represented by the Formula:

X¹-(L)_(a)-X²

[0025] wherein;

[0026] a is 0 or 1;

[0027] X¹ is a peptide comprising an amino acid sequence correspondingto the sequence of residues n+1 through J;

[0028] X² is a peptide comprising an amino acid sequence correspondingto the sequence of residues 1 through n;

[0029] n is an integer ranging from 1 to J−1; and

[0030] L is a linker.

[0031] In the formula above the constituent amino acids residues ofhuman flt3 ligand are numbered sequentially 1 through J from the aminoto the carboxyl terminus. A pair of adjacent amino acids within thisprotein may be numbered n and n+1 respectively where n is an integerranging from 1 to J−1. The residue n+1 becomes the new N-terminus of thenew flt3 receptor agonist and the residue n becomes the new C-terminusof the new flt3 receptor agonist.

[0032] The present invention relates to novel flt3 receptor agonists ofthe following formula:ThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArg SEQ ID NO:145                            10                            20GluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAsp                           30                            40GluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeu                           50                            60LysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHis                           70                            80PheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsn                           90                            100IleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThr                           110                           120ArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuPro                           130                           140ProProTrpSerProArgProLeuGluAlaThrAlaProThrAlaProGlnProProLeu                           150                           160LeuLeuLeuLeuLeuLeuProValGlyLeuLeuLeuLeuAlaAlaAlaTrpCysLeuHis                           170                           180TrpGlnArgThrArgArgArgThrProArgProGlyGluGlnValProProValProSer                           190                           200ProGlnAspLeuLeuLeuValGluHis                         209

[0033] wherein the N-terminus is joined to the C-terminus directly orthrough a linker capable of joining the N-terminus to the C-terminus andhaving new C- and N-termini at amino acids; 28-29 29-30 30-31 31-3232-33 34-35 36-37 37-38 38-39 39-40 40-41 41-42 42-43 64-65 65-66 66-6786-87 87-88 88-89 89-90 90-91 91-92 92-93 93-94 94-95 95-96 96-97 97-9898-99  99-100 100-101 101-102 102-103

[0034] respectively; and

[0035] additionally said flt3 receptor agonist polypeptide can beimmediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine-⁻¹)

[0036] A preferred embodiment is human flt3 receptor agonistpolypeptide, comprising a modified flt3 ligand amino acid sequence ofthe Formula:ThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArg SEQ ID NO:144                            10                            20GluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAsp                           30                            40GluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeu                           50                            60LysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHis                           70                            80PheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsn                           90                            100IleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThr                           110                           120ArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeu                           130

[0037] wherein the N-terminus is joined to the C-terminus directly orthrough a linker capable of joining the N-terminus to the C-terminus andhaving new C- and N-termini at amino acids; 28-29 29-30 30-31 31-3232-33 34-35 36-37 37-38 38-39 39-40 40-41 41-42 42-43 64-65 65-66 66-6786-87 87-88 88-89 89-90 90-91 91-92 92-93 93-94 94-95 95-96 96-97 97-9898-99  99-100 100-101 101-102 102-103

[0038] respectively; and

[0039] additionally said flt3 receptor agonist polypeptide can beimmediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹)

[0040] The more preferred breakpoints at which new C-terminus andN-terminus can be made are 36-37, 37-38, 38-39, 39-40, 40-41, 41-42,42-43, 64-65, 65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92,92-93, 93-94, 95,-96, 96-97, 97-98, 99-100 and 100-101

[0041] The most preferred breakpoints at which new C-terminus 15 andN-terminus can be made are; 39-40, 65-66, 89-90, 99-100 and 100-101.

[0042] The flt3 receptor agonists of the present invention may containamino acid substitutions, deletions and/or insertions. It is alsointended that the flt3 receptor agonists of the present invention mayalso have amino acid deletions at either/or both the N- and C-termini ofthe original protein and or deletions from the new N- and/or C-terminiof the sequence rearranged proteins in the formulas shown above.

[0043] The flt3 receptor agonists of the present invention may containamino acid substitutions, deletions and/or insertions.

[0044] A preferred embodiment of the present invention the linker (L)joining the N-terminus to the C-terminus is a polypeptide selected fromthe group consisting of: GlyGlyGlySer; SEQ ID NO: 38GlyGlyGlySerGlyGlyGlySer; SEQ ID NO: 39GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer; SEQ ID NO: 40SerGlyGlySerGlyGlySer; SEQ ID NO: 41 GluPheGlyAsnMet; SEQ ID NO: 42GluPheGlyGlyAsnMet; SEQ ID NO: 43 GluPheGlyGlyAsnGlyGlyAsnMet; SEQ IDNO: 44 GlyGlySerAspMetAlaGly; SEQ ID NO: 45 SerGlyGlyAsnGly; SEQ ID NO:46 SerGlyGlyAsnGlySerGlyGlyAsnGly; SEQ ID NO: 47SerGlyGlyAsnGlySerGlyGlyAsnGlySerGlyGlyAsnGly; SEQ ID NO: 48SerGlyGlySerGlySerGlyGlySerGly; SEQ ID NO: 49SerGlyGlySerGlySerGlyGlySerGlySerGlyGlySerGly; SEQ ID NO: 50GlyGlyGlySerGlyGly; SEQ ID NO: 51 GlyGlyGlySerGlyGlyGly; SEQ ID NO: 52GlyGlyGlySerGlyGlyGlySerGlyGly; SEQ ID NO: 53GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly; SEQ ID NO: 54GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGly; SEQ ID NO: 55GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly; SEQ ID NO: 56GlyGlySerGly GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly; SEQ IDNO: 148 GlyGlySerGlyGlyGlySerGlyGlyGlySerGlyProProProTrpSerProArgProLeuGlyAlaThrAlaProThrAlaGly; SEQ ID NO: 149GlnProProLeu ProProProTrpSerProArgProLeuGlyAlaThrAlaProThr; SEQ ID NO:150 and ValGluThrValPheHisArgValSerGlnAspGlyLeuLeuThrSer. SEQ ID NO: 151

[0045] The present invention also encompasses recombinant human flt3receptor agonists co-administered or sequentially with one or moreadditional colony stimulating factors (CSF) including, cytokines,lymphokines, interleukins, hematopoietic growth factors which includebut are not limited to GM-CSF, G-CSF, c-mpl ligand (also known as TPO orMGDF), M-CSF, erythropoietin (FLT3), IL-1, IL-4, IL-2, IL-3, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, human growthhormone, B-cell growth factor, B-cell differentiation factor, eosinophildifferentiation factor and stem cell factor (SCF) also known as steelfactor or c-kit ligand (herein collectively referred to as “factors”).These co-administered mixtures may be characterized by having the usualactivity of both of the peptides or the mixture may be furthercharacterized by having a biological or physiological activity greaterthan simply the additive function of the presence of the flt3 receptoragonists or the second colony stimulating factor alone. Theco-administration may also provide an enhanced effect on the activity oran activity different from that expected by the presence of the flt3ligand or the second colony stimulating factor. The co-administrationmay also have an improved activity profile which may include reductionof undesirable biological activities associated with native human flt3ligand. In addition to the list above, IL-3 variants taught in WO94/12639 and WO 94/12638 fusion protein taught in WO 95/21197, and WO95/21254 G-CSF receptor agonists disclosed in WO 97/12977, c-mplreceptor agonists disclosed in WO 97/12978, IL-3 receptor agonistsdisclosed in WO 97/12979 and multi-functional receptor agonists taughtin WO 97/12985 can be co-administered with the polypeptides of thepresent invention. As used herein “IL-3 variants” refer to IL-3 variantstaught in WO 94/12639 and WO 94/12638. As used herein “fusion proteins”refer to fusion protein taught in WO 95/21197, and WO 95/21254. As usedherein “G-CSF receptor agonists” refer to G-CSF receptor agonistsdisclosed in WO 97/12978. As used herein “c-mpl receptor agonists” referto c-mpl receptor agonists disclosed in WO 97/12978. As used herein“IL-3 receptor agonists” refer to IL-3 receptor agonists disclosed in WO97/12979. As used herein “multi-functional receptor agonists” refer tomulti-functional receptor agonists taught in WO 97/12985.

[0046] In addition, it is envisioned that in vitro uses would includethe ability to stimulate bone marrow and blood cell activation andgrowth before the expanded cells are infused into patients. Anotherintended use is for the production of dendritic cells both in vivo andex vivo.

BRIEF DESCRIPTION OF THE FIGURES

[0047]FIG. 1 schematically illustrates the sequence rearrangement of aprotein. The N-terminus (N) and the C-terminus (C) of the native proteinare joined through a linker, or joined directly. The protein is openedat a breakpoint creating a new N-terminus (new N) and a new C-terminus(new-C) resulting in a protein with a new linear amino acid sequence. Arearranged molecule may be synthesized de novo as linear molecule andnot go through the steps of joining the original N-terminus and theC-terminus and opening of the protein at the breakpoint.

[0048]FIG. 2 shows a schematic of Method I, for creating new proteins inwhich the original N-terminus and C-terminus of the native protein arejoined with a linker and different N-terminus and C-terminus of theprotein are created. In the example shown the sequence rearrangementresults in a new gene encoding a protein with a new N-terminus createdat amino acid 97 of the original protein, the original C-terminus (a.a.174) joined to the amino acid 11 (a.a. 1-10 are deleted) through alinker region and a new C-terminus created at amino acid 96 of theoriginal sequence.

[0049]FIG. 3 shows a schematic of Method II, for creating new proteinsin which the original N-terminus and C-terminus of the native proteinare joined without a linker and different N-terminus and C-terminus ofthe protein are created. In the example shown the sequence rearrangementresults in a new gene encoding a protein with a new N-terminus createdat amino acid 97 of the original protein, the original C-terminus (a.a.174) joined to the original N-terminus and a new C-terminus created atamino acid 96 of the original sequence.

[0050]FIG. 4 shows a schematic of Method III, for creating new proteinsin which the original N-terminus and C-terminus of the native proteinare joined with a linker and different N-terminus and C-terminus of theprotein are created. In the example shown the sequence rearrangementresults in a new gene encoding a protein with a new N-terminus createdat amino acid 97 of the original protein, the original C-terminus (a.a.174) joined to amino acid 1 through a linker region and a new C-terminuscreated at amino acid 96 of the original sequence.

[0051]FIG. 5a and 5 b shows the DNA sequence encoding the 209 amino acidmature form of flt3 ligand from Lyman et al. (Oncogene 11:1165-1172,1995).

[0052]FIG. 6 shows the DNA sequence encoding the 134 amino acid solubleform of flt3 ligand from Lyman et al. (Oncogene 11:1165-1172, 1995).

[0053]FIG. 7 shows the bioactivity of the flt3 receptor agonistspMON32320 and pMON32321 compared to recombinant native flt3 (Genzyme)and pMON30237 (1-134 form of the flt3 ligand expressed by mammalian cell(BHK) culture) in the MUTZ-2 cell proliferation assay. MT=mocktransfection.

DETAILED DESCRIPTION OF THE INVENTION

[0054] Flt3 receptor agonists of the present invention may be useful inthe treatment of diseases characterized by decreased levels ofhematopoietic cells.

[0055] A flt3 receptor agonist may be useful in the treatment orprevention of hematopoietic disorders. Many drugs may cause bone marrowsuppression or hematopoietic deficiencies. Examples of such drugs areAZT, DDI, alkylating agents and anti-metabolites used in chemotherapy,antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycinand sulfa drugs, phenothiazones, tranquilizers such as meprobamate,analgesics such as aminopyrine and dipyrone, anti-convulsants such asphenytoin or carbamazepine, antithyroids such as propylthiouracil andmethimazole and diuretics. flt3 receptor agonists may be useful inpreventing or treating the bone marrow suppression or hematopoieticdeficiencies which often occur in patients treated with these drugs.

[0056] Hematopoietic deficiencies may also occur as a result of viral,microbial or parasitic infections, burns and as a result of treatmentfor renal disease or renal failure, e.g., dialysis. The present peptidemay be useful in treating such hematopoietic deficiency.

[0057] Another aspect of the present invention provides plasmid DNAvectors for use in the method of expression of these novel flt3 receptoragonists. These vectors contain the novel DNA sequences described abovewhich code for the novel polypeptides of the invention. Appropriatevectors which can transform host cells capable of expressing the flt3receptor agonists include expression vectors comprising nucleotidesequences coding for the flt3 receptor agonists joined totranscriptional and translational regulatory sequences which areselected according to the host cells used. Vectors incorporatingmodified sequences as described above are included in the presentinvention and are useful in the production of the modified flt3 receptoragonist polypeptides. The vector employed in the method also containsselected regulatory sequences in operative association with the DNAcoding sequences of the invention and capable of directing thereplication and expression thereof in selected host cells.

[0058] As another aspect of the present invention, there is provided anovel method for producing the novel family of human flt3 receptoragonists. The method of the present invention involves culturingsuitable cells or cell line, which has been transformed with a vectorcontaining a DNA sequence coding for expression of the novel flt3receptor agonist polypeptide. Suitable cells or cell lines may includevarious strains of bacteria such as E. coli, yeast, mammalian cells, orinsect cells may be utilized as host cells in the method of the presentinvention.

[0059] Other aspects of the present invention are methods andtherapeutic compositions for treating the conditions referred to above.Such compositions comprise a therapeutically effective amount of one ormore of the flt3 receptor agonists of the present invention in a mixturewith a pharmaceutically acceptable carrier. This composition can beadministered either parenterally, intravenously or subcutaneously. Whenadministered, the therapeutic composition for use in this invention ispreferably in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such a parenterally acceptableprotein solution, having due regard to pH, isotonicity, stability andthe like, is within the skill of the art.

[0060] The dosage regimen involved in a method for treating theabove-described conditions will be determined by the attending physicianconsidering various factors which modify the action of drugs, e.g. thecondition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. Generally,a daily regimen may be in the range of 0.5-150 μg/kg of non-glycosylatedflt3 receptor agonists protein per kilogram of body weight. Dosageswould be adjusted relative to the activity of a given receptor agonistand it would not be unreasonable to note that dosage regimens mayinclude doses as low as 0.1 microgram and as high as 1 milligram perkilogram of body weight per day. In addition, there may exist specificcircumstances where dosages of flt3 receptor agonist would be adjustedhigher or lower than the range of 0.5-150 micrograms per kilogram ofbody weight. These include co-administration with other CSF or growthfactors; co-administration with chemotherapeutic drugs and/or radiation;the use of glycosylated flt3 receptor agonists; and variouspatient-related issues mentioned earlier in this section. As indicatedabove, the therapeutic method and compositions may also includeco-administration with other human factors. A non-exclusive list ofother appropriate hematopoietins, CSFs and interleukins for simultaneousor serial co-administration with the polypeptides of the presentinvention includes GM-CSF, G-CSF, c-mpl ligand (also known as TPO orMGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, human growthhormone, B-cell growth factor, B-cell differentiation factor, eosinophildifferentiation factor and stem cell factor (SCF) also known as steelfactor or c-kit ligand (herein collectively referred to as “factors”),or combinations thereof. In addition to the list above, IL-3 variantstaught in WO 94/12639 and WO 94/12638 fusion protein taught in WO95/21197, and WO 95/21254 G-CSF receptor agonists disclosed in WO97/12977, c-mpl receptor agonists disclosed in WO 97/12978, IL-3receptor agonists disclosed in WO 97/12979 and multi-functional receptoragonists taught in WO 97/12985 can be co-administered with thepolypeptides of the present invention.

[0061] The flt3 receptor agonists of the present invention may be usefulin the mobilization of hematopoietic progenitors and stem cells inperipheral blood. Peripheral blood derived progenitors have been shownto be effective in reconstituting patients in the setting of autologousmarrow transplantation. Hematopoietic growth factors, including G-CSFand GM-CSF, have been shown to enhance the number of circulatingprogenitors and stem cells in the peripheral blood. This has simplifiedthe procedure for peripheral stem cell collection and dramaticallydecreased the cost of the procedure by decreasing the number of pheresisrequired. The flt3 receptor agonist of the present invention may beuseful in mobilization of stem cells and further enhance the efficacy ofperipheral stem cell transplantation.

[0062] The flt3 receptor agonists of the present invention may also beuseful in the ex vivo expansion of hematopoietic progenitors. Colonystimulating factors (CSFs), such as G-CSF, have been administered alone,co-administered with other CSFs, or in combination with bone marrowtransplants subsequent to high dose chemotherapy to treat theneutropenia and which is often the result of such treatment. However theperiod of severe neutropenia may not be totally eliminated. The myeloidlineage, which is comprised of monocytes (macrophages), granulocytes(including neutrophils) and megakaryocytes, is critical in preventinginfections and bleeding which can be life-threatening. Neutropenia mayalso be the result of disease, genetic disorders, drugs, toxins,radiation and many therapeutic treatments such as conventional oncologytherapy.

[0063] Bone marrow transplants have been used to treat this patientpopulation. However, several problems are associated with the use ofbone marrow to reconstitute a compromised hematopoietic systemincluding: 1) the number of stem cells in bone marrow or other tissues,such as spleen or peripheral blood, is limited, 2) Graft Versus HostDisease, 3) graft rejection and 4) possible contamination with tumorcells. Stem cells and progenitor cells make up a very small percentageof the nucleated cells in the bone marrow, spleen and peripheral blood.It is clear that a dose response exists such that a greater number ofmultipotential hematopoietic progenitors will enhance hematopoieticrecovery. Therefore, the in vitro expansion of stem cells should enhancehematopoietic recovery and patient survival. Bone marrow from anallogeneic donor has been used to provide bone marrow for transplant.However, Graft Versus Host Disease and graft rejection limit bone marrowtransplantation even in recipients with HLA-matched sibling donors. Analternative to allogeneic bone marrow transplants is autologous bonemarrow transplants. In autologous bone marrow transplants, some of thepatient's own marrow is harvested prior to myeloablative therapy, e.g.high dose chemotherapy, and is transplanted back into the patientafterwards. Autologous transplants eliminate the risk of Graft VersusHost Disease and graft rejection. However, autologous bone marrowtransplants still present problems in terms of the limited number ofstems cells in the marrow and possible contamination with tumor cells.The limited number of multipotential hematopoietic progenitors may beovercome by ex-vivo expansion of the multipotential hematopoieticprogenitors. In addition, stem cells can be specifically isolated basedon the presence of specific surface antigens such as CD34+ in order todecrease tumor cell contamination of the marrow graft.

[0064] The following patents contain further details on separating stemcells, CD34+ cells, culturing the cells with hematopoietic factors, theuse of the cells for the treatment of patients with hematopoieticdisorders and the use of hematopoietic factors for cell expansion andgene therapy.

[0065] U.S. Pat. No. 5,061,620 relates to compositions comprising humanhematopoietic stem cells provided by separating the stem cells fromdedicated cells.

[0066] U.S. Pat. No. 5,199,942 describes a method for autologoushematopoietic cell transplantation comprising: (1) obtaininghematopoietic progenitor cells from a patient; (2) ex-vivo expansion ofcells with a growth factor selected from the group consisting of IL-3,flt3 ligand, c-kit ligand, GM CSF, IL-1, GM-CSF/IL-3 fusion protein andcombinations thereof; (3) administering cellular preparation to apatient.

[0067] U.S. Pat. No. 5,240,856 relates to a cell separator that includesan apparatus for automatically controlling the cell separation process.

[0068] WO 91/16116 describes devices and methods for selectivelyisolating and separating target cells from a mixture of cells.

[0069] WO 91/18972 describes methods for in vitro culturing of bonemarrow, by incubating suspension of bone marrow cells, using a hollowfiber bioreactor.

[0070] WO 92/18615 relates to a process for maintaining and expandingbone marrow cells, in a culture medium containing specific mixtures ofcytokines, for use in transplants.

[0071] WO 93/08268 describes a method for selectively expanding stemcells, comprising the steps of (a) separating CD34+ stem cells fromother cells and (b) incubating the separated cells in a selectivemedium, such that the stem cells are selectively expanded.

[0072] WO 93/18136 describes a process for in vitro support of mammaliancells derived from peripheral blood.

[0073] WO 93/18648 relates to a composition comprising human neutrophilprecursor cells with a high content of myeloblasts and promyelocytes fortreating genetic or acquired neutropenia.

[0074] WO 94/08039 describes a method of enrichment for humanhematopoietic stem cells by selection for cells which express c-kitprotein.

[0075] WO 94/11493 describes a stem cell population that are CD34+ andsmall in size, which are isolated using a counterflow elutriationmethod.

[0076] WO 94/27698 relates to a method combining immunoaffinityseparation and continuous flow centrifugal separation for the selectiveseparation of a nucleated heterogeneous cell population from aheterogeneous cell mixture.

[0077] WO 94/25848 describes a cell separation apparatus for collectionand manipulation of target cells.

[0078] The long term culturing of highly enriched CD34+ precursors ofhematopoietic progenitor cells from human bone marrow in culturescontaining IL-1α, IL-3, IL-6 or GM-CSF is discussed in Brandt et al (J.Clin. Invest. 86:932-941, 1990).

[0079] One aspect of the present invention provides a method forselective ex-vivo expansion of stem cells. The term “stem cell” refersto the multipotential hematopoietic cells as well as early myeloidprogenitor and precursors cells which can be isolated from bone marrow,spleen or peripheral blood. The term “expansion” refers to theproliferation and differentiation of the cells. The present inventionprovides a method for selective ex-vivo expansion of stem cells,comprising the steps of; (a) separating stem cells from other cells, (b)culturing the separated stem cells with a selective medium whichcontains a flt3 receptor agonist and optionally a second colonystimulating factor, and (c) harvesting the cultured stems cells. Stemcells, as well as committed progenitor cells destined to becomeneutrophils, erythrocytes, platelets, etc., may be distinguished frommost other cells by the presence or absence of particular progenitormarker antigens, such as CD34, that are present on the surface of thesecells and/or by morphological characteristics. The phenotype for ahighly enriched human stem cell fraction is reported as CD34+, Thy-1+and lin−, but it is to be understood that the present invention is notlimited to the expansion of this stem cell population. The CD34+enriched human stem cell fraction can be separated by a number ofreported methods, including affinity columns or beads, magnetic beads orflow cytometry using antibodies directed to surface antigens such as theCD34+. Further, physical separation methods such as counterflowelutriation may be used to enrich hematopoietic progenitors. The CD34+progenitors are heterogeneous, and may be divided into severalsub-populations characterized by the presence or absence ofco-expression of different lineage associated cell surface associatedmolecules. The most immature progenitor cells do not express any knownlineage associated markers, such as HLA-DR or CD38, but they may expressCD90(thy-1). Other surface antigens such as CD33, CD38, CD41, CD71,HLA-DR or c-kit can also be used to selectively isolate hematopoieticprogenitors. The separated cells can be incubated in selected medium ina culture flask, sterile bag or in hollow fibers. Various colonystimulating factors may be utilized in order to selectively expandcells. Representative factors that have been utilized for ex-vivoexpansion of bone marrow include, c-kit ligand, IL-3, G-CSF, GM-CSF,IL-1, IL-6, IL-11, flt3 ligand or combinations thereof. Theproliferation of the stem cells can be monitored by enumerating thenumber of stem cells and other cells, by standard techniques (e.g.hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent toincubation.

[0080] Several methods for ex-vivo expansion of stem cells have beenreported utilizing a number of selection methods and expansion usingvarious colony stimulating factors including c-kit ligand (Brandt etal., Blood 83:1507-1514, 1994; McKenna et al., Blood 86:3413-3420,1995), IL-3 (Brandt et al., Blood 83:1507-1514, 1994; Sato et al., Blood82:3600-3609, 1993), G-CSF (Sato et al., Blood 82:3600-3609, 1993),GM-CSF (Sato et al., Blood 82:3600-3609, 1993), IL-1 (Muench et al.,Blood 81:3463-3473, 1993), IL-6 (Sato et al., Blood 82:3600-3609, 1993),IL-11 (Lemoli et al., Exp. Hem. 21:1668-1672, 1993; Sato et al., Blood82:3600-3609, 1993), flt3 ligand (McKenna et al., Blood 86:3413 3420,1995) and/or combinations thereof (Brandt et al., Blood 83:1507 1514,1994; Haylock et al., Blood 80:1405-1412, 1992, Koller et al.,Biotechnology 11:358-363, 1993; Lemoli et al., Exp. Hem. 21:1668-1672,1993), McKenna et al., Blood 86:3413-3420, 1995; Muench et al., Blood81:3463-3473, 1993; Patchen et al., Biotherapy 7:13-26, 1994; Sato etal., Blood 82:3600-3609, 1993; Smith et al., Exp. Hem. 21:870-877, 1993;Steen et al., Stem Cells 12:214-224, 1994; Tsujino et al., Exp. Hem.21:1379-1386, 1993). Among the individual colony stimulating factors,hIL-3 has been shown to be one of the most potent in expandingperipheral blood CD34+ cells (Sato et al., Blood 82:3600-3609, 1993;Kobayashi et al., Blood 73:1836-1841, 1989). However, no single factorhas been shown to be as effective as the combination of multiplefactors. The present invention provides methods for ex vivo expansionthat utilize novel flt3 receptor agonists.

[0081] Another aspect of the invention provides methods of sustainingand/or expanding hematopoietic precursor cells which includesinoculating the cells into a culture vessel which contains a culturemedium that has been conditioned by exposure to a stromal cell line suchas HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105,1995) that has been supplemented with a flt3 receptor agonist of thepresent invention.

[0082] It is also envisioned that uses of flt3 receptor agonists of thepresent invention would include blood banking applications, where theflt3 receptor agonists are given to a patent to increase the number ofblood cells and blood products are removed from the patient, prior tosome medical procedure, and the blood products are stored and transfusedback into the patient after the medical procedure. Additionally, it isenvisioned that uses of flt3 receptor agonists would include giving theflt3 receptor agonists to a blood donor prior to blood donation toincrease the number of blood cells, thereby allowing the donor to safelygive more blood.

[0083] Another projected clinical use of growth factors has been in thein vitro activation of hematopoietic progenitors and stem cells for genetherapy. Due to the long life-span of hematopoietic progenitor cells andthe distribution of their daughter cells throughout the entire body,hematopoietic progenitor cells are good candidates for ex vivo genetransfection. In order to have the gene of interest incorporated intothe genome of the hematopoietic progenitor or stem cell one needs tostimulate cell division and DNA replication. Hematopoietic stem cellscycle at a very low frequency which means that growth factors may beuseful to promote gene transduction and thereby enhance the clinicalprospects for gene therapy. Potential applications of gene therapy(review Crystal, Science 270:404-410, 1995) include; 1) the treatment ofmany congenital metabolic disorders and immunodeficiencies (Kay and Woo,Trends Genet. 10:253-257, 1994), 2) neurological disorders (Friedmann,Trends Genet. 10:210-214, 1994), 3) cancer (Culver and Blaese, TrendsGenet. 10:174-178, 1994) and 4) infectious diseases (Gilboa and Smith,Trends Genet. 10:139-144, 1994).

[0084] There are a variety of methods, known to those with skill in theart, for introducing genetic material into a host cell. A number ofvectors, both viral and non-viral have been developed for transferringtherapeutic genes into primary cells. Viral based vectors include; 1)replication deficient recombinant retrovirus (Boris-Lawrie and Temin,Curr. Opin. Genet. Dev. 3:102-109, 1993; Boris-Lawrie and Temin, Annal.New York Acad. Sci. 716:59-71, 1994; Miller, Current Top. Microbiol.Immunol. 158:1-24, 1992) and replication-deficient recombinantadenovirus (Berkner, BioTechniques 6:616-629, 1988; Berkner, CurrentTop. Microbiol. Immunol. 158:39-66, 1992; Brody and Crystal, Annal. NewYork Acad. Sci. 716:90-103, 1994). Non-viral based vectors includeprotein/DNA complexes (Cristiano et al., PNAS USA. 90:2122-2126, 1993;Curiel et al., PNAS USA 88:8850-8854, 1991; Curiel, Annal. New YorkAcad. Sci. 716:36-58, 1994), electroporation and liposome mediateddelivery such as cationic liposomes (Farhood et al., Annal. New YorkAcad. Sci. 716:23-35, 1994).

[0085] The present invention provides an improvement to the existingmethods of expanding hematopoietic cells, into which new geneticmaterial has been introduced, in that it provides methods utilizing flt3receptor agonists that may have improved biological activity and/orphysical properties.

[0086] Another intended use of the flt-3 receptor agonists of thepresent invention is for the generation of larger numbers of dendriticcells, from precursors, to be used as adjuvants for immunization.Dendritic cells play a crucial role in the immune system. They are theprofessional antigen-presenting cells most efficient in the activationof resting T cells and are the major antigen-presenting cells foractivation of naïve T cells in vivo and, thus, for initiation of primaryimmune responses. They efficiently internalize, process and presentsoluble tumor-specific antigens (Ag). Dendritic cells have the uniquecapacity to cluster naïve T cells and to respond to Ag encounter byrapid up-regulation of the expression of major histocompatabilitycomplex (MHC) and co-stimulatory molecules, the production of cytokinesand migration towards lymphatic organs. Since dendritic cells are ofcentral importance for sensitizing the host against a neoantigen forCD4-dependent immune responses, they may also play a crucial role in thegeneration and regulation of tumor immunity.

[0087] Dendritic cells originate from a bone marrow CD34+ precursorcommon to granulocytes and macrophages, and the existence of a separatedendritic cell colony-forming unit (CFU-DC) that give rise to puredendritic cell colonies has been established in humans. In addition, apost-CFU CD14+ intermediate has been described with the potential todifferentiate along the dendritic cell or the macrophage pathway underdistinct cytokine conditions. This bipotential precursor is present inthe bone marrow, cord blood and peripheral blood. Dendritic cells can beisolated by the cell specific marker, CD83, which is expressed on maturedendritic cells, to delineate the maturation of cultured dendriticcells.

[0088] Dendritic cells based strategies provide a method for enhancingimmune response against tumors and infectious agents. AIDS is anotherdisease for which dendritic cell based therapies can be used, sincedendritic cells can play a major role in promoting HIV-1 replication. Animmunotherapy requires the generation of dendritic cells from cancerpatients, their in vitro exposure to tumor Ag, derived from surgicallyremoved tumor masses, and reinjection of these cells into the tumorpatients. Relatively crude membrane preparations of tumor cells willsuffice as sources of tumor antigen, avoiding the necessity formolecular identification of the tumor antigen. The tumor antigen mayalso be synthetic peptides, carbohydrates, or nucleic acid sequences. Inaddition, concomitant administration of cytokines such as the flt-3receptor agonists of the present invention may further facilitate theinduction of tumor immunity. It is foreseen that the immunotherapy canbe in an in vivo setting, wherein the flt-3 receptor agonists of thepresent invention is administered to a patient, having a tumor, alone orwith other hematopoietic growth factors to increase the number ofdendritic cells and endogenous tumor antigen is presented on thedendritic cells. It is also envisioned that in vivo immunotherapy can bewith exogenous antigen. It is also envisioned that the immunotherapytreatment may include the mobilization of dendritic cell precursors ormature dendritic, by administering the flt-3 receptor agonists of thepresent invention alone or with other hematopoietic growth factors tothe patient, removing the dendritic cell precursors or mature dendriticcells from the patient, exposing the dendritic cells to antigen andreturning the dendritic cells to the patient. Furthermore, the dendriticcells that have been removed can be cultured ex vivo with the flt-3receptor agonists of the present invention alone or with otherhematopoietic growth factors to increase the number of dendritic cellsprior to exposure to antigen. Dendritic cells based strategies alsoprovide a method for reducing the immune response in auto-immunediseases.

[0089] Studies on dendritic cells have been greatly hampered bydifficulties in preparing the cells in sufficient numbers and in areasonably pure form. In an ex-vivo cell expansion setting,granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumornecrosis factor-α (TNF-α) cooperate in the ex vivo generation ofdendritic cells from hematopoietic progenitors (CD34+ cells) retrievedfrom bone marrow, cord blood, or peripheral blood and flk-2/flt-3 ligandand c-kit ligand (stem cell factor [SCF]) synergize to enhance theGM-CSF plus TNF-α induced generation of dendritic cells (Siena, S. etal. Experimental Hematology 23:1463-1471, 1995). Also provide is amethod of ex vivo expansion of dendritic cell precursors or maturedendritic cells using the flt-3 receptor agonists of the presentinvention to provide sufficient quantities of dendritic cells forimmunotherapy.

Determination of the Linker

[0090] The length of the amino acid sequence of the linker can beselected empirically or with guidance from structural information, or byusing a combination of the two approaches.

[0091] When no structural information is available, a small series oflinkers can be prepared for testing using a design whose length isvaried in order to span a range from 0 to 50 Å and whose sequence ischosen in order to be consistent with surface exposure (hydrophilicity,Hopp & Woods, Mol. Immunol. 20: 483-489, 1983; Kyte & Doolittle, J. Mol.Biol. 157:105-132, 1982; solvent exposed surface area, Lee & Richards,J. Mol. Biol. 55:379-400, 1971) and the ability to adopt the necessaryconformation without deranging the configuration of the flt3 receptoragonist (conformationally flexible; Karplus & Schulz,Naturwissenschaften 72:212-213, (1985). Assuming an average oftranslation of 2.0 to 3.8 Å per residue, this would mean the length totest would be between 0 to 30 residues, with 0 to 15 residues being thepreferred range. Exemplary of such an empirical series would be toconstruct linkers using a cassette sequence such as Gly-Gly-Gly-Serrepeated n times, where n is 1, 2, 3 or 4. Those skilled in the art willrecognize that there are many such sequences that vary in length orcomposition that can serve as linkers with the primary considerationbeing that they be neither excessively long nor short (cf., Sandhu,Critical Rev. Biotech. 12: 437-462, 1992); if they are too long, entropyeffects will likely destabilize the three-dimensional fold, and may alsomake folding kinetically impractical, and if they are too short, theywill likely destabilize the molecule because of torsional or stericstrain.

[0092] Those skilled in the analysis of protein structural informationwill recognize that using the distance between the chain ends, definedas the distance between the c-alpha carbons, can be used to define thelength of the sequence to be used, or at least to limit the number ofpossibilities that must be tested in an empirical selection of linkers.They will also recognize that it is sometimes the case that thepositions of the ends of the polypeptide chain are ill-defined instructural models derived from x-ray diffraction or nuclear magneticresonance spectroscopy data, and that when true, this situation willtherefore need to be taken into account in order to properly estimatethe length of the linker required. From those residues whose positionsare well defined are selected two residues that are close in sequence tothe chain ends, and the distance between their c-alpha carbons is usedto calculate an approximate length for a linker between them. Using thecalculated length as a guide, linkers with a range of number of residues(calculated using 2 to 3.8 Å per residue) are then selected. Theselinkers may be composed of the original sequence, shortened orlengthened as necessary, and when lengthened the additional residues maybe chosen to be flexible and hydrophilic as described above; oroptionally the original sequence may be substituted for using a seriesof linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:38)cassette approach mentioned above; or optionally a combination of theoriginal sequence and new sequence having the appropriate total lengthmay be used.

Determination of the Amino and Carboxyl Termini of flt3 ReceptorAgonists

[0093] Sequences of flt3 receptor agonists capable of folding tobiologically active states can be prepared by appropriate selection ofthe beginning (amino terminus) and ending (carboxyl terminus) positionsfrom within the original polypeptide chain while using the linkersequence as described above. Amino and carboxyl termini are selectedfrom within a common stretch of sequence, referred to as a breakpointregion, using the guidelines described below. A novel amino acidsequence is thus generated by selecting amino and carboxyl termini fromwithin the same breakpoint region. In many cases the selection of thenew termini will be such that the original position of the carboxylterminus immediately preceded that of the amino terminus. However, thoseskilled in the art will recognize that selections of termini anywherewithin the region may function, and that these will effectively lead toeither deletions or additions to the amino or carboxyl portions of thenew sequence.

[0094] It is a central tenet of molecular biology that the primary aminoacid sequence of a protein dictates folding to the three-dimensionalstructure necessary for expression of its biological function. Methodsare known to those skilled in the art to obtain and interpretthree-dimensional structural information using x-ray diffraction ofsingle protein crystals or nuclear magnetic resonance spectroscopy ofprotein solutions. Examples of structural information that are relevantto the identification of breakpoint regions include the location andtype of protein secondary structure (alpha and 3-10 helices, paralleland anti-parallel beta sheets, chain reversals and turns, and loops;Kabsch & Sander, Biopolymers 22: 2577-2637, 1983; the degree of solventexposure of amino acid residues, the extent and type of interactions ofresidues with one another (Chothia, Ann. Rev. Biochem. 53:537-572; 1984)and the static and dynamic distribution of conformations along thepolypeptide chain (Alber & Mathews, Methods Enzymol. 154: 511-533,1987). In some cases additional information is known about solventexposure of residues; one example is a site of post-translationalattachment of carbohydrate which is necessarily on the surface of theprotein. When experimental structural information is not available, oris not feasible to obtain, methods are also available to analyze theprimary amino acid sequence in order to make predictions of proteintertiary and secondary structure, solvent accessibility and theoccurrence of turns and loops. Biochemical methods are also sometimesapplicable for empirically determining surface exposure when directstructural methods are not feasible; for example, using theidentification of sites of chain scission following limited proteolysisin order to infer surface exposure (Gentile & Salvatore, Eur. J.Biochem. 218:603-621, 1993). Thus using either the experimentallyderived structural information or predictive methods (e.g., Srinivisan &Rose Proteins: Struct., Funct. & Genetics, 22: 81-99, 1995) the parentalamino acid sequence is inspected to classify regions according towhether or not they are integral to the maintenance of secondary andtertiary structure. The occurrence of sequences within regions that areknown to be involved in periodic secondary structure (alpha and 3-10helices, parallel and anti-parallel beta sheets) are regions that shouldbe avoided. Similarly, regions of amino acid sequence that are observedor predicted to have a low degree of solvent exposure are more likely tobe part of the so-called hydrophobic core of the protein and should alsobe avoided for selection of amino and carboxyl termini. In contrast,those regions that are known or predicted to be in surface turns orloops, and especially those regions that are known not to be requiredfor biological activity, are the preferred sites for location of theextremes of the polypeptide chain. Continuous stretches of amino acidsequence that are preferred based on the above criteria are referred toas a breakpoint region. TABLE 1 OLIGONUCLEOTIDES NCOFLTCTGACCATGGCNACCCAGGACTGCTCCTTCCAA; SEQ ID NO: 57 HIND160ACTGAAGCTTAGGGCTGACACTGCAGCTCCAG; SEQ ID NO: 58 HIND165ACTGAAGCTTACAGGGTTGAGGAGTCGGGCTG; SEQ ID NO: 59 FL23ForGACTGCCATGGCNACYCAGGAYTGYTCYTTYCAACACAGCCCCATC; SEQ ID NO: 60 FH3AForGACTGCCATGGCNACYCAGGAYTGYTCYTTYCAACACAGCCCCATC; SEQ ID NO: 61 SCF.REVTGTCCAAACTCATCAATGTATC; SEQ ID NO: 62 39FORCATGGCCATGGCCGACGAGGAGCTCTGCGGGGGCCTCT; SEQ ID NO: 63 39REVGCTAGAAGCTTACTGCAGGTTGGAGGCCACGGTGAC; SEQ ID NO: 64 65FORCATGGCCATGGCCTCCAAGATGCAAGGCTTGCTGGAGC; SEQ ID NO: 65 65REVGCTAGAAGCTTACCCAGCGACAGTCTTGAGCCGCTC; SEQ ID NO: 66 89FORCATGGCCATGGCCCCCCCCAGCTGTCTTCGCTTCGT; SEQ ID NO: 67 89REVGCTAGAAGCTTAGGGCTGAAAGGCACATTTGGTGACA; SEQ ID NO: 68 L5ACCCTGTCTGGCGGCAACGGCACCCAGGACTGCTCCTTCCAAC; SEQ ID NO: 69 L10AGCGGTAACGGCAGTGGAGGTAATGGCACCCAGGACTGCTCCTTCCAAC; SEQ ID NO: 70 L15AACGGCAGTGGTGGCAATGGGAGCGGCGGAAATGGAACCCAGGACTGCTCCT; SEQ ID NO: 71TCCAAC L5B GTGCCGTTGCCGCCAGACAGGGTTGAGGAGTCGGGCTG; SEQ ID NO: 72 L10BATTACCTCCACTGCCGTTACCGCCTGACAGGGTTGAGGAGTCGGGCTG; SEQ ID NO: 73 L15BGCTCCCATTGCCACCACTGCCGTTACCTCCAGACAGGGTTGAGGA; SEQ ID NO: 74 GTCGGGCTGL15C GATGAGGATCCGGTGGCAATGGGAGCGGCGGAAATGGAACCCAGG; SEQ ID NO: 75ACTGCTCCTTCCACC L15D GATGACGGATCCGTTACCTCCAGACAGGGTTGAGGAGTCGGGCTG; SEQID NO: 76 L15E GATGACGGATCCGGAGGTAATGGCACCCAGGACTGCTCCTTCCAAC; SEQ IDNO: 77 339FOR2 GACTGCCATGGCCGACGAGGAGCTCTGCG; SEQ ID NO: 78 339REV2GACTCAAGCTTACTGCAGGTTGGAGGCC; SEQ ID NO: 79 339-10FOR3GACTCGGGATCCGGAGGTTCTGGCACCCAGGACTGCTCC; SEQ ID NO: 80 339-15FOR2GACTGGGATCCGGTGGCAGTGGGAGCGGCGGATCTGGAACC; SEQ ID NO: 81 339REV3GACTTGGGATCCACTACCTCCAGACAGGGTTGAGGAGTC; SEQ ID NO: 82 FLN3ACTGACGGATCCACCGCCCAGGGTTGAGGAGTCGGGCTG; SEQ ID NO: 83 FLN7ACTGACGGATCCACCTCCTGACCCACCGCCCAGGGTTGAGGAGTCGGGCTG; SEQ ID NO: 84 FLN11ACTGACGGATCCACCTCCTGACCCACCTCCTGACCCACCGCCCAG; SEQ ID NO: 85GGTTGAGGAGTCGGGCTG C-term ACGTAAAGCTTACAGGGTTGAGGAGTCG; SEQ ID NO: 86FLC3 GTCAGTGGATCCGGAGGTACCCAGGACTGCTCCTTCCAAC; SEQ ID NO: 87 FLC4GTCAGTGGATCCGGAGGTGGCACCCAGGACTGCTCCTTCCAAC; SEQ ID NO: 88 FLC10GTCAGTGGATCCGGAGGTGGCTCAGGGGGAGGTAGTGGTACCCAG; SEQ ID NO: 89GACTGCTCCTTCCAAC Flt36GTTGCCATGGCNTCNAAYCTGCARGAYGARGARCTGTGCGGGGGCCTCTGG; SEQ ID NO: 90CGGCTG Flt37 GTTGCCATGGCNAAYCTGCARGAYGARGARCTGTGYGGGGGCCTCTGGCG; SEQ IDNO: 91 GCTGGTC Flt38GTTGCCATGGCNCTGCARGAYGARGARCTGTGYGGYGGCCTCTGGCGGCTG; SEQ ID NO: 92GTCCTG Flt39 GTTGCCATGGCNCARGAYGARGARCTGTGYGGYGGYCTCTGGCGGCTGGTC; SEQ IDNO: 93 CTGGCA Flt40 GTTGCCATGGCNGAYGARGARCTGTGYGGYGGYCTCTGGCGGCTGGTCCTG;SEQ ID NO: 94 GCACAG Flt41GTTGCCATGGCNGARGARCTGTCYGGYGGYCTCTGGCGGCTGGTCCTGGCA; SEQ ID NO: 95CAGCGC Flt42 GTTGCCATGGCNGARCTGTGYGGYGGYCTGTGGCGYCTGGTCCTGGCACAG; SEQ IDNO: 96 CGCTGG Flt43 GTTGCCATGGCNCTGTGYGGYGGYCTGTGGCGYCTGGTCCTGGCACAGCGC;SEQ ID NO: 97 TGGATG 36REV TATGCAAGCTTAGGCCACGGTGACTGGGTA; SEQ ID NO: 9837REV TATGCAAGCTTAGGAGGCCACGGTGACTGG; SEQ ID NO: 99 38REVTATGCAAGCTTAGTTGGAGGCCACGGTGAC; SEQ ID NO: 100 39REVTATGCAAGCTTACAGGTTGGAGGCCACGGT; SEQ ID NO: 101 40REVTATGCAAGCTTACTGCAGGTTGGAGGCCAC; SEQ ID NO: 102 41REVTATGCAAGCTTAGTCCTGCAGGTTGGAGGC; SEQ ID NO: 103 42REVTATGCAAGCTTACTCGTCCTGCAGGTTGGA; SEQ ID NO: 104 43REVTATGCAAGCTTACTCCTCGTCCTGCAGGTT; SEQ ID NO: 105

[0095] TABLE 2 DNA SEQUENCES pMON30237.seqGCCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGC; SEQ ID NO: 106TGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGCGCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTC AGCCC pMON30238.seqGCCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGC; SEQ ID NO: 107TGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTC AGCCCGACTCCTCAACCCTGpMON30239.seq GCCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGC; SEQ IDNO: 108 TGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCC CGACTCCTCAACCCTGpMON32329.seq GGAACTCAGGATTGTTCTTTCCAACACAGCCCCATCTCCTCCGACTTCGC; SEQ IDNO: 109 TGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTC AGCCC pMON32330.seqGGTACCCAGGATTGTTCTTTCCAACACAGCCCCATCTCCTCCGACTTCGC; SEQ ID NO: 110TGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTC AGCCCGACTCCTCAACCCTGpMON32341.seq GCCACTCAGGACTGTTCTTTCCAACACAGCCCCATCTCCTCCGACTTCGC; SEQ IDNO: 111 TGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTC AGCCC pMON32342.seqGCCACTCAGGACTGCTCTTTTCAACACAGCCCCATCTCCTCCGACTTCGC; SEQ ID NO: 112TGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTC AGCCCGACTCCTCAACCCTGpMON32320.seq GCCGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ IDNO: 113 CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGAGGTAACGGATCCGGTGGCAATGGGAGCGGCGGAAATGGAACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTG GCCTCCAACCTGCAGpMON32321.seq GCCGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ IDNO: 114 CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCAGGCGGTAACGGCAGTGGAGGTAATGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAG pMON32322.seqGCCGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ ID NO: 115CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGCGGCAACGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAG pMON32323.seqGCCTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTT; SEQ ID NO: 116TGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGAGGTAACGGATCCGGTGGCAATGGGAGCGGCGGAAATGGAACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAA4ATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTC AAGACTGTCGCTGGGGCGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTT; SEQ ID NO: 117TGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGAGGTAACGGATCCGGAGGTAATGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGG pMON32325.seqGCCTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTT; SEQ ID NO: 118TGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGCGGCAACGGCACGCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGG pMON32326.seqGCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCT; SEQ ID NO: 119GCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGAGGTAACGGCAGTGGTGGCAATGGGAGCGGTGGAAATGGAACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAA TGTGCCTTTCAGCCCpMON32327.seq GCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCT; SEQ IDNO: 120 GCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCAGGCGGTAACGGCAGTGGAGGTAATGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAkATGTGCCTTTCAGCCC pMON32328.seqGCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCT; SEQ ID NO: 121GCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGCGGCAACGGCACGCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCC pMON32348.seqGCCGACGAGGACCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ ID NO: 122CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTCCAGCGCGTCAACACGGAGATACACTTTGTCACCAAATCTCCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGAGGTAGTGGATCCGGAGGTTCTGGCAACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAG pMON32350.seqGCCGACGAGGAGCTCTGCGGGGGCCTCTGGCGCCTGGTCCTGGCACAGCG; SEQ ID NO: 123CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTCTGGAGGTACTGGATCCGGTGGCAGTGCGAGCGGCGGATCTCGAACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTC GCCTCCAACCTGCAGFLT3N.seq CCATGGCCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGAC; SEQ ID NO:124 TTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGCAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTCGATGGAGCGGCTCAACACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCCCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGCAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGATCC FLT3C.seqGGATCCGGAGGTACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTC; SEQ ID NO: 125CGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTCGGTCCAAGATGCAAGCCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTCGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTAAGCTT FLT7N.seqCCATGGCCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGAC; SEQ ID NO: 126TTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCCCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGATCC FLT4C.seqGGATCCGGAGGTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTC; SEQ ID NO: 127CTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGTAAGCTT FLT11N.seqCCATGGCCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGAC; SEQ ID NO: 128TTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCCCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGCTGCGTCAGGAGGTGGGTCAG GAGGTGGATCCFLT10C.seq GGATCCGGAGGTGGCTCAGGGGGAGGTAGTGGTACCCAGGACTGCTCCTT; SEQ IDNO: 129 CCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTG TAAGCTT pMON32365.seqGCCGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ ID NO: 130CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGATCCGGAGGTACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTCACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAG pMON32366.seqGCCGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ ID NO: 131CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTCCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGATCCGGAGGTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAG pMON32367.seqGCCGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ ID NO: 132CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATCTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCACCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGATCCGGAGGTACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTCGCCTCCAACCTGCAG pMON32368.seqGCCGACGAGGAGCTCTGCGGGGCCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ ID NO: 133CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTCGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGATCCGGAGGTGGCTCAGGGGGAGGTAGTGGTACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCCACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCC AACCTGCAGpMON32369.seq GCCGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCG; SEQ IDNO: 134 CTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCACCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGGTCAGGAGGTGGATCCCGAGGTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTG GCCTCCAACCTGCAGpMON32370.seq GCCGACGAGGAGCTCTGCGGGGGCCTCTCGCGGCTGGTCCTGGCACAGCG; SEQ IDNO: 135 CTGGATGGAGCGGCTCAAGACTCTCGCTGGCTCCAAGATGCAAGGCTTGCTGGAGCGCGTCAACACGGAGATACACTTTCTCACCAAATGTCCCTTTCAGCCCCCCCCCAGCTGCCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGGTCAGGAGGTGGATCCGGAGGTGGCTCAGGGGGAGGTAGTGGTACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAG pMON35712.seqGCCGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGG; SEQID NO: 136CTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGGTCAGGAGGTGGATCCGGAGGTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTT CAApMON35713.seqGCCGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGC; SEQID NO: 137TGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGGTCAGGAGGTGGATCCGGAGGTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCCCTCTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACC GTGpMON35714.seqGCCGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACC; SEQID NO: 138AAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGGTCAGGAGGTGGATCCGGAGCTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAG ACTpMON35715.seqGCCTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCC; SEQID NO: 139TTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGGTCAGGAGGTGGATCCGGAGGTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCT GGGpMON35716.seqGCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAG; SEQID NO: 140CAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGGTCAGGAGGTGGATCCGGAGGTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAG CCCpMON 35717.seqGCCCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTG; SEQID NO: 142AAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGGTGGGTCAGGAGGTGGATCCGGAGGTGGCACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGACCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATCTCCCTTTCAGCCCCCCCCCAGCTGT CTTpMON 35718.seqGCCACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATC; SEQID NO: 143ACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGGGCGGTGGGTCAGGAGCTGGGTCACGAGGTGGATCCGGAGGTGGCACCCACGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCCGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTC CAG

[0096] TABLE 3 PROTEIN SEQUENCES pMON30237.pepAlaThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuS;SEQ ID NO: 1erAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyAlaLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnPro pMON30238.pepAlaThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuS;SEQ ID NO: 2erAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeupMON30239.pepAlaThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuS;SEQ ID NO: 3erAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnGluThrSerGluGinLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeu pMON32329.pepGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuS;SEQ ID NO: 4erAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnPro pMON32330.pepGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuS;SEQ ID NO: 5erAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeupMON32341.pepAlaThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuS;SEQ ID NO: 6erAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnPro pMON32342.pepAlaThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuS;SEQ ID NO: 7erAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeupMON32320.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 8hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlySerGlyGlyAsnGlySerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32321.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 9hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeubysProTrpIleThrArgGlnAsnPheSermrgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlySerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32322.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 10hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32323.pepAlaSerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheG;SEQ ID NO: 11lnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlySerGlyGlyAsnGlySerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGly pMON32324.pepAlaSerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheG;SEQ ID NO: 12lnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlySerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGly pMON32325.pepAlaSerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheG;SEQ ID NO: 13lnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGly pMON32326.pepAlaProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSeroluGlnL;SEQ ID NO: 14euValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlySerGlyGlyAsnGlySerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnPro pMON32327.pepAlaProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnL;SEQ ID NO: 15euValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlySerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnPro pMON32328.pepAlaProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnL;SEQ ID NO: 16euValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlyAsnGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnPro pMON32348.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 17hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlySerGlySerGlyGlySerGlySerGlyGlySerGlyThrGlnAspCysSerPheclnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32350.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 18hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuSerGlyGlySerGlySerGlyGlySerGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln FLT3N.pepMetAlaThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluL;SEQ ID NO: 19euSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySer FLT3C.pepGlySerGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleA;SEQ ID NQ: 20rgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeu FLT7N.pepMetAlaThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluL;SEQ ID NO: 21euSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySer FLT4C.pepGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysI;SEQ ID NO: 22leArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeu FLT11N.pepMetAlaThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluL;SEQ ID NO: 23euSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySer FLT10C.pepGlySerGlyGlyGlySerGlyGlyGlySerGlyThrGlnAspCysSerPheGlnHisSerProIleSerS;SEQ ID NO: 24erAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeu pMON32365.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 25hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32366.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 26hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32367.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 27hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32368.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 28hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32369.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 29hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGln pMON32370.pepAlaAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysT;SEQ ID NO: 30hrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnpMON35712.pepAlaAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuV;SEQ ID NO: 31alLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGln pMON35713.pepAlaAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpM;SEQ ID NO: 32etGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrVal pMON35714.pepAlaValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysC;SEQ ID NO: 33ysAlaPheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThr pMON35715.pepAlaSerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheG;SEQ ID NO: 34lnProProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGly pMON35716.pepAlaProProSerCysLeuArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnL;SEQ ID NO: 35euValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnPro pMON35717.pepAlaArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysP;SEQ ID NO: 36roTrpIleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeu pMON35718.pepAlaThrAsnIleSerArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrpIleThrA;SEQ ID NO: 37rgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnProAspSerSerThrLeuGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAlaPheGlnProProProSerCysLeuArgPheValGln

Materials and Methods Recombinant DNA methods

[0097] Unless noted otherwise, all specialty chemicals were obtainedfrom Sigma Co., (St. Louis, Mo.). Restriction endonucleases and T4 DNAligase were obtained from New England Biolabs (Beverly, Mass.) orBoehringer Mannheim (Indianapolis, Ind.).

Transformation of E. coli Strains

[0098]E. coli strains, such as DH5α™ (Life Technologies, Gaithersburg,Md.) and TG1 (Amersham Corp., Arlington Heights, Ill.) are used fortransformation of ligation reactions and are the source of plasmid DNAfor transfecting mammalian cells. E. coli strains, such as MON105 andJM101, can be used for expressing the flt3 receptor agonist of thepresent invention in the cytoplasm or periplasmic space.

[0099] MON105 ATCC#55204: F-, lamda-, IN(rrnD, rrE)1, rpoD+, rpoH358

[0100] DH5α™: F-, phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR,recA1, endA1, hsdR17(rk−, mk+), phoA, supE44lamda-, thi-1, gyrA96, relA1

[0101] TG1: delta(lac-pro), supE, thi-1, hsdD5/F′(traD36, proA+B+,lacIq, lacZdeltaM15)

[0102] DH5α™ Subcloning efficiency cells are purchased as competentcells and are ready for transformation using the manufacturer'sprotocol, while both E. coli strains TG1 and MON105 are renderedcompetent to take up DNA using a CaCl₂ method. Typically, 20 to 50 mL ofcells are grown in LB medium (1% Bacto-tryptone, 0.5% Bacto-yeastextract, 150 mM NaCl) to a density of approximately 1.0 optical densityunit at 600 nanometers (OD600) as measured by a Baush & Lomb Spectronicspectrophotometer (Rochester, N.Y.). The cells are collected bycentrifugation and resuspended in one-fifth culture volume of CaCl₂solution (50 mM CaCl₂, 10 mM Tris-Cl, pH 7.4) and are held at 4° C. for30 minutes. The cells are again collected by centrifugation andresuspended in one-tenth culture volume of CaCl₂ solution. Ligated DNAis added to 0.2 mL of these cells, and the samples are held at 4° C. for1 hour. The samples are shifted to 42° C. for two minutes and 1 mL of LBis added prior to shaking the samples at 37° C. for one hour. Cells fromthese samples are spread on plates (LB medium plus 1.5% Bacto-agar)containing either ampicillin (100 micrograms/mL, ug/mL) when selectingfor ampicillin-resistant transformants, or spectinomycin (75 ug/mL) whenselecting for spectinomycin-resistant transformants. The plates areincubated overnight at 37° C. Single colonies are picked, grown in LBsupplemented with appropriate antibiotic for 6-16 hours at 37° C. withshaking. Colonies are picked and inoculated into LB plus appropriateantibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and aregrown at 37° C. while shaking. Before harvesting the cultures, 1 ul ofcells are analyzed by PCR for the presence of a flt3 receptor agonistgene. The PCR is carried out using a combination of primers that annealto the flt3 receptor agonist gene and/or vector. After the PCR iscomplete, loading dye is added to the sample followed by electrophoresisas described earlier. A gene has been ligated to the vector when a PCRproduct of the expected size is observed.

Methods for Creation of Genes With New N-terminus/C-terminus

[0103] Method I. Creation of Genes With New N-terminus/C-terminus WhichContain a Linker Region.

[0104] Genes with new N-terminus/C-terminus which contain a linkerregion separating the original C-terminus and N-terminus can be madeessentially following the method described in L. S. Mullins, et al J.Am. Chem. Soc. 116, 5529-5533 (1994). Multiple steps of polymerase chainreaction (PCR) amplifications are used to rearrange the DNA sequenceencoding the primary amino acid sequence of the protein. The steps areillustrated in FIG. 2.

[0105] In the first step, the primer set (“new start” and “linkerstart”) is used to create and amplify, from the original gene sequence,the DNA fragment (“Fragment Start”) that contains the sequence encodingthe new N-terminal portion of the new protein followed by the linkerthat connects the C-terminal and N-terminal ends of the originalprotein. In the second step, the primer set (“new stop” and “linkerstop”) is used to create and amplify, from the original gene sequence,the DNA fragment (“Fragment Stop”) that encodes the same linker as usedabove, followed by the new C-terminal portion of the new protein. The“new start” and “new stop” primers are designed to include theappropriate restriction enzyme recognition sites which allow cloning ofthe new gene into expression plasmids. Typical PCR conditions are onecycle 95° C. melting for two minutes; 25 cycles 94° C. denaturation forone minute, 50° C. annealing for one minute and 72° C. extension for oneminute; plus one cycle 72° C. extension for seven minutes. A PerkinElmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains100 pmole of each primer and one ug of template DNA; and 1×PCR buffer,200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaqDNA polymerase and 2 mM MgCl₂. PCR reactions are performed in a Model480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, Conn.).

[0106] “Fragment Start” and “Fragment Stop”, which have complementarysequence in the linker region and the coding sequence for the two aminoacids on both sides of the linker, are joined together in a third PCRstep to make the full-length gene encoding the new protein. The DNAfragments “Fragment Start” and “Fragment Stop” are resolved on a 1% TAEgel, stained with ethidium bromide and isolated using a Qiaex GelExtraction kit (Qiagen). These fragments are combined in equimolarquantities, heated at 70° C. for ten minutes and slow cooled to allowannealing through their shared sequence in “linker start” and “linkerstop”. In the third PCR step, primers “new start” and “new stop” areadded to the annealed fragments to create and amplify the full-lengthnew N-terminus/C-terminus gene. Typical PCR conditions are one cycle 95°C. melting for two minutes; 25 cycles 94° C. denaturation for oneminute, 60° C. annealing for one minute and 72° C. extension for oneminute; plus one cycle 72° C. extension for seven minutes. A PerkinElmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains100 pmole of each primer and approximately 0.5 ug of DNA; and 1×PCRbuffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 unitsAmpliTaq DNA polymerase and 2 mM MgCl₂. PCR reactions are purified usinga Wizard PCR Preps kit (Promega).

[0107] Method II. Creation of Genes With New N-terminus/C-terminusWithout a Linker Region.

[0108] New N-terminus/C-terminus genes without a linker joining theoriginal N-terminus and C-terminus can be made using two steps of PCRamplification and a blunt end ligation. The steps are illustrated inFIG. 3. In the first step, the primer set (“new start” and “P-bl start”)is used to create and amplify, from the original gene sequence, the DNAfragment (“Fragment Start”) that contains the sequence encoding the newN-terminal portion of the new protein. In the second step, the primerset (“new stop” and “P-bl stop”) is used to create and amplify, from theoriginal gene sequence, the DNA fragment (“Fragment Stop”) that containsthe sequence encoding the new C-terminal portion of the new protein. The“new start” and “new stop” primers are designed to include appropriaterestriction sites which allow cloning of the new gene into expressionvectors. Typical PCR conditions are one cycle 95° C. melting for twominutes; 25 cycles 94° C. denaturation for one minute, 50° C. annealingfor 45 seconds and 72° C. extension for 45 seconds. Deep Vent polymerase(New England Biolabs) is used to reduce the occurrence of overhangs inconditions recommended by the manufacturer. The “P-bl start” and “P-blstop” primers are phosphorylated at the end to aid in the subsequentblunt end ligation of “Fragment Start” and “Fragment Stop” to eachother. A 100 ul reaction contained 150 pmole of each primer and one ugof template DNA; and 1× Vent buffer (New England Biolabs), 300 uM dGTP,300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit Deep Vent polymerase.PCR reactions are performed in a Model 480 DNA thermal cycler (PerkinElmer Corporation, Norwalk, Conn.). PCR reaction products are purifiedusing a Wizard PCR Preps kit (Promega).

[0109] The primers are designed to include appropriate restrictionenzyme recognition sites which allow for the cloning of the new geneinto expression vectors. Typically “Fragment Start” is designed tocreate a NcoI restriction site, and “Fragment Stop” is designed tocreate a HindIII restriction site. Restriction digest reactions arepurified using a Magic DNA Clean-up System kit (Promega). FragmentsStart and Stop are resolved on a 1% TAE gel, stained with ethidiumbromide and isolated using a Qiaex Gel Extraction kit (Qiagen). Thesefragments are combined with and annealed to the ends of the ˜3800 basepair NcoI/HindIII vector fragment of pMON3934 by heating at 50° C. forten minutes and allowed to slow cool. The three fragments are ligatedtogether using T4 DNA ligase (Boehringer Mannheim). The result is aplasmid containing the full-length new N-terminus/C-terminus gene. Aportion of the ligation reaction is used to transform E. coli strainDH5α cells (Life Technologies, Gaithersburg, Md.). Plasmid DNA ispurified and sequence confirmed as below.

[0110] Method III. Creation of New N-terminus/C-terminus Genes byTandem-Duplication Method

[0111] New N-terminus/C-terminus genes can be made based on the methoddescribed in R. A. Horlick, et al Protein Eng. 5:427-431 (1992).Polymerase chain reaction (PCR) amplification of the newN-terminus/C-terminus genes is performed using a tandemly duplicatedtemplate DNA. The steps are illustrated in FIG. 4.

[0112] The tandemly-duplicated template DNA is created by cloning andcontains two copies of the gene separated by DNA sequence encoding alinker connecting the original C- and N-terminal ends of the two copiesof the gene. Specific primer sets are used to create and amplify afull-length new N terminus/C-terminus gene from the tandemly-duplicatedtemplate DNA. These primers are designed to include appropriaterestriction sites which allow for the cloning of the new gene intoexpression vectors. Typical PCR conditions are one cycle 95° C. meltingfor two minutes; 25 cycles 94° C. denaturation for one minute, 50° C.annealing for one minute and 72° C. extension for one minute; plus onecycle 72° C. extension for seven minutes. A Perkin Elmer GeneAmp PCRCore Reagents kit (Perkin Elmer Corporation, Norwalk, Conn.) is used. A100 ul reaction contains 100 pmole of each primer and one ug of templateDNA; and 1×PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uMdCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl₂. PCR reactionsare performed in a Model 480 DNA thermal cycler (Perkin ElmerCorporation, Norwalk, Conn.). PCR reactions are purified using a WizardPCR Preps kit (Promega).

DNA Isolation and Characterization

[0113] Plasmid DNA can be isolated by a number of different methods andusing commercially available kits known to those skilled in the art. Afew such methods are shown herein. Plasmid DNA is isolated using thePromega Wizard™ Miniprep kit (Madison, Wis.), the Qiagen QIAwell Plasmidisolation kits (Chatsworth, Calif.) or Qiagen Plasmid Midi kit. Thesekits follow the same general procedure for plasmid DNA isolation.Briefly, cells are pelleted by centrifugation (5000×g), plasmid DNAreleased with sequential NaOH/acid treatment, and cellular debris isremoved by centrifugation (10000×g). The supernatant (containing theplasmid DNA) is loaded onto a column containing a DNA-binding resin, thecolumn is washed, and plasmid DNA eluted with TE. After screening forthe colonies with the plasmid of interest, the E. coli cells areinoculated into 50-100 mLs of LB plus appropriate antibiotic forovernight growth at 37° C. in an air incubator while shaking. Thepurified plasmid DNA is used for DNA sequencing, further restrictionenzyme digestion, additional subcloning of DNA fragments andtransfection into mammalian, E. coli or other cells.

Sequence Confirmation

[0114] Purified plasmid DNA is resuspended in dH₂O and quantitated bymeasuring the absorbance at 260/280 nm in a Bausch and Lomb Spectronic601 UV spectrometer. DNA samples are sequenced using ABI PRISM™DyeDeoxy™ terminator sequencing chemistry (Applied Biosystems Divisionof Perkin Elmer Corporation, Lincoln City, Calif.) kits (Part Number401388 or 402078) according to the manufacturers suggested protocolusually modified by the addition of 5% DMSO to the sequencing mixture.Sequencing reactions are performed in a Model 480 DNA thermal cycler(Perkin Elmer Corporation, Norwalk, Conn.) following the recommendedamplification conditions. Samples are purified to remove excess dyeterminators with Centri-Sep™ spin columns (Princeton Separations,Adelphia, N.J.) and lyophilized. Fluorescent dye labeled sequencingreactions are resuspended in deionized formamide, and sequenced ondenaturing 4.75% polyacrylamide-8M urea gels using an ABI Model 373Aautomated DNA sequencer. Overlapping DNA sequence fragments are analyzedand assembled into master DNA contigs using Sequencher DNA analysissoftware (Gene Codes Corporation, Ann Arbor, Mich.).

Expression of flt3 Receptor Agonists in Mammalian Cells Mammalian CellTransfection/Production of Conditioned Media

[0115] The BHK-21 cell line can be obtained from the ATCC (Rockville,Md.). The cells are cultured in Dulbecco's modified Eagle media(DMEM/high-glucose), supplemented to 2 mM (mM) L-glutamine and 10% fetalbovine serum (FBS). This formulation is designated BHK growth media.Selective media is BHK growth media supplemented with 453 units/mLhygromycin B (Calbiochem, San Diego, Calif.). The BHK-21 cell line waspreviously stably transfected with the HSV transactivating protein VP16,which transactivates the IE110 promoter found on the plasmid pMON3359(See Hippenmeyer et al., Bio/Technology, pp.1037-1041, 1993). The VP16protein drives expression of genes inserted behind the IE110 promoter.BHK-21 cells expressing the transactivating protein VP16 are designatedBHK-VP16. The plasmid pMON1118 (See Highkin et al., Poultry Sci., 70:970-981, 1991) expresses the hygromycin resistance gene from the SV40promoter. A similar plasmid is available from ATCC, pSV2-hph.

[0116] BHK-VP16 cells are seeded into a 60 millimeter (mm) tissueculture dish at 3×10⁵ cells per dish 24 hours prior to transfection.Cells are transfected for 16 hours in 3 mL of “OPTIMEM”™ (Gibco-BRL,Gaithersburg, Md.) containing 10 ug of plasmid DNA containing the geneof interest, 3 ug hygromycin resistance plasmid, pMON1118, and 80 ug ofGibco-BRL “LIPOFECTAMINE”™ per dish. The media is subsequently aspiratedand replaced with 3 mL of growth media. At 48 hours post-transfection,media from each dish is collected and assayed for activity (transientconditioned media). The cells are removed from the dish by trypsin-EDTA,diluted 1:10 and transferred to 100 mm tissue culture dishes containing10 mL of selective media. After approximately 7 days in selective media,resistant cells grow into colonies several millimeters in diameter. Thecolonies are removed from the dish with filter paper (cut toapproximately the same size as the colonies and soaked in trypsin/EDTA)and transferred to individual wells of a 24 well plate containing 1 mLof selective media. After the clones are grown to confluence, theconditioned media is re-assayed, and positive clones are expanded intogrowth media.

Expression of flt3 Receptor Agonists in E. coli

[0117]E. coli strain MON105 or JM101 harboring the plasmid of interestare grown at 37° C. in M9 plus casamino acids medium with shaking in aair incubator Model G25 from New Brunswick Scientific (Edison, N.J.).Growth is monitored at OD600 until it reaches a value of 1, at whichtime nalidixic acid (10 milligrams/mL) in 0.1 N NaOH is added to a finalconcentration of 50 μg/mL. The cultures are then shaken at 37° C. forthree to four additional hours. A high degree of aeration is maintainedthroughout culture period in order to achieve maximal production of thedesired gene product. The cells are examined under a light microscopefor the presence of inclusion bodies (IB). One mL aliquots of theculture are removed for analysis of protein content by boiling thepelleted cells, treating them with reducing buffer and electrophoresisvia SDS-PAGE (see Maniatis et al. Molecular Cloning: A LaboratoryManual, 1982). The culture is centrifuged (5000×g) to pellet the cells.

[0118] Additional strategies for achieving high-level expression ofgenes in E. coli can be found in Savvas, C. M. (Microbiological Reviews60;512-538, 1996).

[0119] Inclusion Body Preparation, Extraction, Refolding, Dialysis, DEAEChromatography, and Characterization of the flt3 Receptor Agonists WhichAccumulate as Inclusion Bodies in E. coli.

[0120] Isolation of Inclusion Bodies:

[0121] The cell pellet from a 330 mL E. coli culture is resuspended in15 mL of sonication buffer (10 mM 2-amino-2-(hydroxymethyl)1,3-propanediol hydrochloride (Tris-HCl), pH 8.0+1 mMethylenediaminetetraacetic acid (EDTA)). These resuspended cells aresonicated using the microtip probe of a Sonicator Cell Disruptor (ModelW-375, Heat Systems-Ultrasonics, Inc., Farmingdale, N.Y.). Three roundsof sonication in sonication buffer followed by centrifugation areemployed to disrupt the cells and wash the inclusion bodies (IB). Thefirst round of sonication is a 3 minute burst followed by a 1 minuteburst, and the final two rounds of sonication are for 1 minute each.

[0122] Extraction and Refolding of Proteins from Inclusion Body Pellets:

[0123] Following the final centrifugation step, the IB pellet isresuspended in 10 mL of 50 mM Tris-HCl, pH 9.5, 8 M urea and 5 mMdithiothreitol (DTT) and stirred at room temperature for approximately45 minutes to allow for denaturation of the expressed protein.

[0124] The extraction solution is transferred to a beaker containing 70mL of 5 mM Tris-HCl, pH 9.5 and 2.3 M urea and gently stirred whileexposed to air at 4° C. for 18 to 48 hours to allow the proteins torefold. Refolding is monitored by analysis on a Vydac (Hesperia, Calif.)C18 reversed phase high pressure liquid chromatography (RP-HPLC) column(0.46×25 cm). A linear gradient of 40% to 65% acetonitrile, containing0.1% trifluoroacetic acid (TFA), is employed to monitor the refold. Thisgradient is developed over 30 minutes at a flow rate of 1.5 mL perminute. Denatured proteins generally elute later in the gradient thanthe refolded proteins.

[0125] Purification:

[0126] Following the refold, contaminating E. coli proteins are removedby acid precipitation. The pH of the refold solution is titrated tobetween pH 5.0 and pH 5.2 using 15% (v/v) acetic acid (HOAc). Thissolution is stirred at 4° C. for 2 hours and then centrifuged for 20minutes at 12,000×g to pellet any insoluble protein.

[0127] The supernatant from the acid precipitation step is dialyzedusing a Spectra/Por 3 membrane with a molecular weight cut off (MWCO) of3,500 daltons. The dialysis is against 2 changes of 4 liters (a 50-foldexcess) of 10 mM Tris-HCl, pH 8.0 for a total of 18 hours. Dialysislowers the sample conductivity and removes urea prior to DEAEchromatography. The sample is then centrifuged (20 minutes at 12,000×g)to pellet any insoluble protein following dialysis.

[0128] A Bio-Rad Bio-Scale DEAE2 column (7×52 mm) is used for ionexchange chromatography. The column is equilibrated in a buffercontaining 10 mM Tris-HCl, pH 8.0. The protein is eluted using a0-to-500 mM sodium chloride (NaCl) gradient, in equilibration buffer,over 45 column volumes. A flow rate of 1 mL per minute is usedthroughout the run. Column fractions (2 mL per fraction) are collectedacross the gradient and analyzed by RP HPLC on a Vydac (Hesperia,Calif.) C18 column (0.46×25 cm). A linear gradient of 40% to 65%acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is employed.This gradient is developed over 30 minutes at a flow rate of 1.5 mL perminute. Pooled fractions are then dialyzed against 2 changes of 4 liters(50-to-500-fold excess) of 10 mM ammonium acetate (NH₄Ac), pH 4.0 for atotal of 18 hours. Dialysis is performed using a Spectra/Por 3 membranewith a MWCO of 3,500 daltons. Finally, the sample is sterile filteredusing a 0.22μm syringe filter (μStar LB syringe filter, Costar,Cambridge, Mass.), and stored at 4 ° C.

[0129] In some cases the folded proteins can be affinity purified usingaffinity reagents such as mAbs or receptor subunits attached to asuitable matrix. Alternatively, (or in addition) purification can beaccomplished using any of a variety of chromatographic methods such as:ion exchange, gel filtration or hydrophobic chromatography or reversedphase HPLC.

[0130] These and other protein purification methods are described indetail in Methods in Enzymology, Volume 182 ‘Guide to ProteinPurification’ edited by Murray Deutscher, Academic Press, San Diego,Calif. (1990).

[0131] Protein Characterization:

[0132] The purified protein is analyzed by RP-HPLC, electrospray massspectrometry, and SDS-PAGE. The protein quantitation is done by aminoacid composition, RP-HPLC, and Bradford protein determination. In somecases tryptic peptide mapping is performed in conjunction withelectrospray mass spectrometry to confirm the identity of the protein.

[0133] Methylcellulose Assay

[0134] This assay reflects the ability of colony stimulating factors tostimulate normal bone marrow cells to produce different types ofhematopoietic colonies in vitro (Bradley et al., Aust. Exp Biol. Sci.44:287-300, 1966), Pluznik et al., J. Cell Comp. Physio 66:319-324,1965).

[0135] Methods

[0136] Approximately 30 mL of fresh, normal, healthy bone marrowaspirate are obtained from individuals following informed consent. Understerile conditions samples are diluted 1:5 with a 1×PBS (#14040.059 LifeTechnologies, Gaithersburg, Md.) solution in a 50 mL conical tube(#25339-50 Corning, Corning Md.). Ficoll (Histopaque 1077 Sigma H-8889)is layered under the diluted sample and centrifuged, 300×g for 30 min.The mononuclear cell band is removed and washed two times in 1×PBS andonce with 1% BSA PBS (CellPro Co., Bothel, Wash.). Mononuclear cells arecounted and CD34+ cells are selected using the Ceprate LC (CD34) Kit(CellPro Co., Bothel, Wash.) column. This fractionation is performedsince all stem and progenitor cells within the bone marrow display CD34surface antigen.

[0137] Cultures are set up in triplicate with a final volume of 1.0 mLin a 35×10 mm petri dish (Nunc#174926). Culture medium is purchased fromTerry Fox Labs. (HCC-4230 medium (Terry Fox Labs, Vancouver, B.C.,Canada) and erythropoietin (Amgen, Thousand Oaks, Calif.) is added tothe culture media. 3,000-10,000 CD34+ cells are added per dish. FLT3receptor agonist proteins, in conditioned media from transfectedmammalian cells or purified from conditioned media from transfectedmammalian cells or E. coli are added to give final concentrationsranging from 0.001 nM to 10 nM. Cultures are resuspended using a 3 ccsyringe and 1.0 mL is dispensed per dish. Control (baseline response)cultures received no colony stimulating factors. Positive controlcultures received conditioned media (PHA stimulated human cells: TerryFox Lab. H2400). Cultures are incubated at 37° C., 5% CO₂ in humidifiedair.

[0138] Hematopoietic colonies which are defined as greater than 50 cellsare counted on the day of peak response (days 10-11) using a Nikoninverted phase microscope with a 40× objective combination. Groups ofcells containing fewer than 50 cells are referred to as clusters.Alternatively colonies can be identified by spreading the colonies on aslide and stained or they can be picked, resuspended and spun ontocytospin slides for staining.

[0139] Human Cord Blood Hemopoietic Growth Factor Assays

[0140] Bone marrow cells are traditionally used for in vitro assays ofhematopoietic colony stimulating factor (CSF) activity. However, humanbone marrow is not always available, and there is considerablevariability between donors. Umbilical cord blood is comparable to bonemarrow as a source of hematopoietic stem cells and progenitors(Broxmeyer et al., PNAS USA 89:4109-113, 1992; Mayani et al., Blood81:3252-3258, 1993). In contrast to bone marrow, cord blood is morereadily available on a regular basis. There is also a potential toreduce assay variability by pooling cells obtained fresh from severaldonors, or to create a bank of cryopreserved cells for this purpose.

[0141] Methods

[0142] Mononuclear cells (MNC) are isolated from cord blood within 24hr. of collection, using a standard density gradient (1.077 g/mLHistopaque). Cord blood MNC have been further enriched for stem cellsand progenitors by several procedures, including immunomagneticselection for CD14−, CD34+ cells; panning for SBA−, CD34+ fraction usingcoated flasks from Applied Immune Science (Santa Clara, Calif.); andCD34+ selection using a CellPro (Bothell, Wash.) avidin column. Eitherfreshly isolated or cryopreserved CD34+ cell enriched fractions are usedfor the assay. Duplicate cultures for each serial dilution of sample(concentration range from 1 pM to 1204 pM) are prepared with 1×104 cellsin 1 ml of 0.9% methylcellulose containing medium without additionalgrowth factors (Methocult H4230 from Stem Cell Technologies, Vancouver,BC.). In some experiments, Methocult H4330 containing erythropoietin(FLT3) was used instead of Methocult H4230, or Stem Cell Factor (SCF),50 ng/mL (Biosource International, Camarillo, Calif.) was added. Afterculturing for 7-9 days, colonies containing >30 cells are counted.

[0143] MUTZ-2 Cell Proliferation Assay

[0144] A cell line such as MUTZ-2, which is a human myeloid leukemiacell line (German Collection of Microorganisms and Cell Cultures, DSMACC 271), can be used to determine the cell proliferative activity offlt3 receptor agonists. MUTZ-2 cultures are maintained with recombinantnative flt3 ligand (20-100 ng/mL) in the growth medium. Eighteen hoursprior to assay set-up, MUTZ-2 cells are washed in IMDM medium (Gibco)three times and are resuspended in IMDM medium alone at a concentrationof 0.5-0.7×10E6 cells/mL and incubated at 37° C. and 5% CO₂ to starvethe cells of flt3 ligand. The day of the assay, standards and flt3receptor agonists are diluted to two fold above desired finalconcentration in assay media in sterile tissue culture treated 96 wellplates. Flt3 receptor agonists and standards are tested in triplicate.50 μl of assay media is loaded into all wells except row A. 75 μl of theflt3 receptor agonists or standards are added to row A and 25 μl takenfrom that row and serial dilutions (1:3) performed on the rest of theplate (rows B through G). Row H remains as a media only control. Thestarved MUTZ-2 cells are washed two times in IMDM medium and resuspendedin 50 μl assay media. 50 μl of cells are added to each well resulting ina final concentration of 0.25×10E6cells/mL. Assay plates containingcells are incubated at 37° C. and 5% CO₂ for 44 hrs. Each well is thenpulsed with 1 μCi/well of tritiated thymidine in a volume of 20 μl forfour hours. Plates are then harvested and counted.

[0145] Transfected Cell Lines:

[0146] Cell lines, such as BHK or the murine pro B cell line Baf/3, canbe transfected with a colony stimulating factor receptor, such as thehuman flt3 receptor which the cell line does not have. These transfectedcell lines can be used to determine the activity of the ligand of whichthe receptor has been transfected.

EXAMPLE 1

[0147] Isolation of cDNA Encoding flt3 Ligand

[0148] Three flt3 ligand clones were amplified from human bone morrowpoly A+ RNA (Clontech) using NCOFLT, HIND160, and HIND165 PCR primers(according to the manufacturer's suggested conditions). These amplifiedPCR products were gel purified and cloned into the BHK expression vectorpMON5723 generating pMON30237 (NCOFLT+HIND160), pMON30238(NCOFLT+HIND165), and a deletion clone pMON30239 (NCOFLT+HIND165). Thedeletion in pMON30239 is of amino acid residues 89 through 106 (thenumbering of the residues is based on the sequence of native flt3 ligandas shown in FIGS. 5a and 5 b).

EXAMPLE 2

[0149] Sequence rearranged flt3 ligand were constructed using severalmethods and linker types. The first set of constructs containing thelinker peptide (SerGlyGlyAsnGly) (SEQ ID NO:46) (where X=1, 2, or 3)with the breakpoints 39/40, 65/66, and 89/90 were made using a two stepPCR process described by Mullins et al. in which the front half and theback half of each final sequence rearranged molecule is made separatelyin the first PCR step, then the paired products of the first reactionstep are combined in a second PCR step and extended in the absence ofexogenous primers.

[0150] For example, to make the three 89/90 breakpoint precursormolecules with the SerGlyGlyAsnGly SEQ ID NO: 46,SerGlyGlyAsnGlySerGlyGlyAsnGly SEQ ID NO: 47, andSerGlyGlyAsnGlySerGlyGlyAsnGlySerGlyGlyAsnGly SEQ ID NO:48 amino acidlinkers (pMON32326, pMON32327 and pMON32328 respectively), six initialPCR products were generated. The following primer pairs were used in thefirst step PCR reaction: a) 89For/L5B; b) 89For/L10B; c) 89For/L15B; d)89Rev/L5A; e) 89Rev/L10A; and f) 89Rev/L15A. The identical approach wasused to make pMON32321 (39/40 breakpoint, primer pairs 39For/L10B and39Rev/L10A) and pMON32325 (65/66 breakpoint, primer pairs 65For/L5B and65Rev/L5A) precursors. Except as noted below, all subsequent PCRreactions utilized the components of the PCR Optimizer Kit (Invitrogen)and amplification conditions according to the manufacturers suggestedprotocol. Reactions were set up as follows: 50 pmole of each primer, 10ul of 5× Buffer B [300 mM Tris-HCl (pH 8.5), 10 MM MgCl₂, 75 mM(NH4)2SO4], 5 U Taq polymerase, and 100 ng of heat denatured DNA (inthis example pMON30238) template were combined, and brought to 45 ulfinal volume with dH₂O. Reactions were pre-incubated for 1-5 minute at80° C., then 5 ul of 10 mM dNTP added to each reaction, and heatdenatured for 2 minutes at 94° C. prior to amplification in a PerkinElmer model 480 DNA thermal cycler. Seven DNA amplification cycles weredone under the following conditions: heat denature for one minute at 94°C., two minutes annealing at 65° C., followed by a three minuteextension at 72° C. Twenty three additional cycles consisting of a oneminute heat denaturation at 94° C. followed by a four minuteannealing/extension at 72° C. were done, followed by a final 7 minuteextension cycle at 72° C. With the exception of pMON32328, the PCRamplification products were run out on a 1.2% TAE agarose gel, and theappropriate size bands (the major amplification product) were excisedand purified using Geneclean II (Bio 101). Samples were resuspended in10 ul dH₂O. The amplification products for pMON32328 were purifieddirectly using a Wizard PCR Clean UP kit (Promega), and DNA eluted in 50ul dH₂O.

[0151] The method to construct the precursors of pMON32322 (39/40breakpoint, primer pairs 39For/L5B and 39Rev/L5A) was modified byincreasing the amount of template to 1 ug, and by changing the PCRamplification conditions as follows: six cycles of 94° C., 1 minute, 65°C. for 2 minute, and 72° C. for 2½ minutes, followed by 15 cycles of 94°C. for 1 minute, 70° C. for 2 minutes, and 72° C. for 2 minutes,followed by a single 72° C. extension cycle for seven minutes.

[0152] The second PCR step utilized the gel-purified precursors from thefirst PCR step as a combination of primer/template as follows: 5 ul eachof each precursor molecule (i.e. for pMON32328 the PCR products fromprimer pairs 89For/L5B and 89Rev/L5A), 10 ul of 5× Buffer B, 5 U of Taqpolymerase, and 24 ul dH₂O. The reactions were heated for five minutesat 80° C., 5 ul of 10 mM dNTP was added, and the reactions heatdenatured for 94° C. for two minutes. DNA amplification conditions wereas follows: 15 cycles of 94° C. for one minute, 69° C. for two minutes,followed then by a three minute extension at 72° C. To allow forcomplete extension, the last cycle was followed by a single extensionstep at 72° C. for seven minutes. The 80 deg incubation time was reducedto two minutes and the number of cycles was decreased to ten cycles forpMON32325 (PCR products 65For/L5B and 65Rev/L5A). PCR reaction productsof the appropriate size were gel purified on a 1.2% TAE agarose gelusing Geneclean II. For pMON32322 (39For/L5B and 39Rev/L5A) theannealing temperature was reduced to 68° C., and the extension timereduced to two minutes. In addition, the PCR product was purified usinga Wizard PCR Clean Up kit (Promega) according to the suppliers suggestedprotocol. The second PCR step was modified for pMON32326 (PCR productsof 89For/L15B and 89Rev/L15A) as follows. Three sets of PCR reactionswere set up identically as above, except for the sample buffer type(either 5× buffer B, D, or J-PCR Optimizer Kit). Composition of buffersD and J differ from buffer B only by pH or [MgCl₂]. The [MgCl₂] forbuffer D is 3.5 mM, whereas the pH of buffer J is 9.5. The protocol wasmodified by increasing the number of PCR cycles 20, and 15 ul aliquotswere withdrawn at the end of cycles 10, 15 and 20. Five uls of eachaliquot timepoint were analyzed for the presence of amplified materialon a 1.2% TBE agarose gel. The remainder of the buffer B, D, and J PCRreaction mixtures were pooled and subsequently purified using the WizardPCR Clean Up Kit protocol. The DNA was eluted in 50 ul dH₂O.

[0153] The purified samples from the second step PCR reaction weredigested with NcoI/HindIII using one of two standardized digestionconditions. For Geneclean II purified samples, 10 ul of DNA weredigested in a 20 ul reaction with 7.5 U each of NcoI/HindIII for twohours at 37° C., and gel purified on a 1.1% TAE agarose gel again withGeneclean II. Ligation-ready samples were resuspended in 10 ul dH₂O. ForpMON32322, 20 ul of sample was digested in a 50 ul reaction volume with20 U each of NcoI and HindIII for 3 hour at 37° C. 0.1 volume 3M NaOAc(pH 5.5) and 2.5 volume of EtOH were added, mixed, and stored at −20° C.overnight. The DNA was recovered by pelleting for 20 minutes at 13,000rpm @ 4° C. in a Sigma Mk 202 microfuge. The DNA pellet was rinsed withchilled 70% EtOH, lyophilized, and resuspended in 10 ul dH₂O.

EXAMPLE 3

[0154] An alternate approach was used to construct pMON32320 (39/40breakpoint, fifteen amino acid linker), pMON32323 (65/66 breakpoint,fifteen AA linker), and pMON32324 (65/66 breakpoint, ten amino acidlinker). New primers (L15C, L15D, L15E) were designed to incorporateBamHI restriction site in the primer that was inframe to allow cloninginto the BamHI site and maintain the proper reading frame. PCR reactionconditions for the first step were performed identically to thatdescribed for pMON32322, except that the following set of primer pairswere used: 65For/L15D and 65Rev/L15E (pMON32324); 39For/L15D and39Rev/L15C (pMON32320); and 65For/L15D and 65Rev/L15C (pMON32323). ThePCR reaction products were purified using a Wizard PCR Clean Up kit asdescribed, and eluted in 50 ul dH₂O. Samples were digested with eitherNcoI/BamHI (39For/L15D and 65For/L15D) or BamHI/HindIII (39Rev/L15C,65Rev/L15C, and 65Rev/L15E). Restriction digests were performed asfollows: 10 ul of purified PCR reaction products, 3 ul of 10× universalrestriction buffer, 15 U of either NcoI or HindIII, 15 U of BamHI, in afinal reaction volume of 30 ul. Reactions were incubated for 90 minutesat 37° C., and the PCR products gel purified on a 1.1% TAE agarose gelusing Geneclean II. Ligation-ready DNA was resuspended in 10 ul dH₂O.

[0155] Inserts were ligated to NcoI/ HindIII digested pMON3977 (BHKmammalian expression vector) that had been treated with shrimp alkalinephosphatase (SAP) either in a three way (pMON32320, pMON32323, orpMON32324) or a two way (pMON32321, pMON32322, pMON32325, pMON32326,pMON32327 and pMON32328) ligation reaction as follows: 2.5 ul of insert(2 ul of each primer pair amplicon for pMON32320, pMON32323, andpMON32324) was added to 50 ng of vector in a ten ul reaction usingstandard ligation conditions. Two ul of each reaction was transformedwith 100 ul of chemically competent DH5α cells (Gibco/BRL) following themanufacturers suggested protocol. Twenty five ul and 200 ul aliquotswere plated out on LB plates containing 50 ug/mL ampicillin andincubated overnight. Isolated colonies were picked and DNA prepared from50 mL overnight cultures using Qiagen DNA midiprep kits. DNA wasquantitated by absorbance at A260/A280, and verified for correct insertsize by agarose gel electrophoresis following digestion of 1 ug templatewith NcoI/HindIII restriction endonucleases. Samples containing insertsof the predicted size were sequenced in both orientations usingvector-specific primers using an automated fluorescent DNA sequencermodel 373A (Perkin Elmer ABI). Sequencing reactions were done in 20 ulreaction volumes using a Perkin Elmer model 480 DNA thermal cycler asfollows: one ug of template, 3.2 pmole primer, 1 ul DMSO, 9.5 ul Taqterminator dyedeoxy premix (Perkin Elmer ABI) were combined, andsubjected to 25 cycles of sequencing amplification as follows: 30seconds at 94° C., 15 second annealing at 50C, followed by a four minuteextension cycle at 60° C. Samples were purified using Centri-Sep spincolumns (Princeton Separations) following the manufacturers suggestedprotocol, lyophilized, and submitted for sequence analysis. Samplescontaining the predicted amino acid sequence were selected for analysisand assigned pMON numbers.

EXAMPLE 4

[0156] A similar approach used to construct pMON32320, pMON32323, andpMON32324 was utilized to introduce the second linker type(SerGlyGlySerGly)X where x=2 or 3, into two sequence rearranged flt3receptor agonists containing the 39/40 breakpoint (pMON32348 and 32350).The primer pairs were as follows: for pMON32348 the combinations of339For2/339Rev3 and 339Rev2/339-10For3 and for pMON32350 thecombinations of 339For2/339Rev3 and 339Rev2/339-15For3 were used tocreate three PCR amplification products. Each PCR amplification was setup as follows: to 100 ng of heat denatured pMON32320, 50 pmole of eachprimer pair, 10 ul of 5× Buffer B, 5 U of Taq polymerase and dH₂O wasadded to a final volume of 45 ul. Reactions were pre-incubated asdescribed before. Fifteen amplification cycles were done under thefollowing conditions: heat denature at 94° C., one minute, followed by atwo minute annealing step at 70° C., and a three minute extension at 72°C. After the last cycle, a single 72 deg extension step of 7 minutes wasdone. The PCR amplification products of primer pairs 339For2/339Rev3,339Rev2/339-10For3, and 339Rev2/339-15For2 were purified using a WizardPCR Clean Up kit (Promega), and eluted in 50 ul dH₂O. NcoI/BamHI digestsfor the 339For2/339Rev3 primer pair as follows: 8 ul of DNA template wasmixed with 2 ul universal restriction buffer and 10 U each of NcoI andBamHI in a 20 ul reaction volume, and incubated for 90 minutes at 37° C.The digestion products was purified using the Geneclean II directpurification protocol, and ligation ready DNA resuspended in 10 ul dH₂O.The restriction digests and subsequent purification for the339Rev2/339-10For3 and 339Rev2/339-15For2 amplification products weredone identically as described for the 339For2/339Rev3 amplicon, exceptthat 10 U of HindIII was substituted for NcoI. Standard ligations weredone by adding to 50 ng NcoI/HindIII/SAP-treated, gel purified pMON3977,0.5 ul 339For2/Rev3 amplicon, 1 ul of either 339Rev2/339-10For3(pMON32348) or 339Rev2/339-15For3 (pMON32350) amplicons, 5U T4 DNAligase, and 1 ul 10× ligase buffer in a 10 ul reaction volume for 60minutes at ambient temperature. Subsequent steps leading to final DNAsequence confirmation were done as described above.

EXAMPLE 5

[0157] A third type of linker, with a variable (GlyGlyGlySer) (SEQ IDNO:38) repeat motif, was incorporated into another set of sequencerearranged flt3 receptor agonists from modularly constructed templates.These linker lengths were;

[0158] 6 AA linker (GlyGlyGlySerGlyGly SEQ ID NO:51),

[0159] 7 AA linker (GlyGlyGlySerGlyGlyGly SEQ ID NO:52),

[0160] 10 AA linker (GlyGlyGlySerGlyGlyGlySerGlyGly SEQ ID NO:53),

[0161] 13 AA linker (GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly SEQ IDNO:54),

[0162] 15 AA linker (GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGly SEQID NO:55) ; and

[0163] 21 AA linker(GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGly GlySerGly SEQ IDNO:56) amino acid residues. These modular templates, each comprising adimer of hflt3 ligand separated by a BamHI-containing linker of uniquelength, were constructed as follows. Six intermediate PLASMID templates,FL3N, FL7N, FL11N, FL3C, FL4C, and FL10C, were constructed by PCR usingpaired primers and pMON30238 as template using cycling conditionssimilar to those employed for pMON32322. Per reaction, 50 pmole of eachprimer was added to 100 ng of heat-denatured template and the reactionsassembled as described for pMON32322. Cycle conditions were as follows:seven cycles of 94° C., one minute; two minutes at 65° C., and 2.5minutes at 72° C.; followed by ten cycles of one minute at 94° C., twominutes at 70° C., and 2.5 minutes at 72° C. A single seven minuteextension at 72° C. completed the cycling reactions. The primer pairsused to construct each intermediate were; N-term/FLN3 (FL3N);N-term/FLN7 (FL7N); N-term/FLN11 (FL11N); C term/FLC3 (FL3C);C-term/FLC4 (FL4C); and C-term/FLC10 (FL10C). The PCR amplificationproducts were purified with Wizard PCR Clean Up kits (Promega) andeluted in 50 ul dH₂O. Purified DNA for the first subset, FL3N, FL7N, andFL11N, were digested with NcoI/BamHI, gel purified as describedpreviously, and ligated to NcoI/BamHI/Sap-treated pSE420 vector DNA(Invitrogen). Intermediate templates of the second subset, FL3C, FL4C,and FL10C, were constructed in an identical manner except HindIII wasutilized instead of NcoI. Subsequent steps leading to final DNA sequenceconfirmation were done as described above.

EXAMPLE 6

[0164] To make the next six templates, the two subsets of intermediatesin pSE420 were digested with either NcoI/BamHI (FL3N, FL7N,FL11N-subset 1) or BamHI/HindIII (FL3C, FL4C, FL10C-subset 2) and gelpurified using Geneclean II as described previously. One intermediateamplicon from each subset were ligated to NcoI/HindIII/SAP-treatedpMON3977 per reaction and transformed in DH5α cells as describedpreviously using the following combinations to generate specific linkerlengths: six AA linker (FL3N and FL3C), seven AA linker (FL3N and FL4C),ten AA linker (FL7N and FL3C), thirteen AA linker (FL3N and FL10C),fifteen AA linker (FL11N and FL4C), and 21 AA linker (FL11N and FL10C).DNA was prepared 50 mL overnight cultures from single colonies from eachof the six combination as described above, analyzed for correct insertsize by NcoI/HindIII restriction analysis, and used as template.

[0165] Primer pairs 39For/39Rev (39/40 breakpoint); 65For/65Rev (65/66breakpoint) and 89For/89Rev (89/90 breakpoint) were used to PCR amplifyeach templates as described for pMON32322, except 75 pmole of eachprimer was used. Amplification conditions were modified as follows: sixcycles of 94° C. for one minute, 2 minutes at 70° C., 2.5 minutes at 72°C.; followed by nine cycles of 94° C. for one minute, and three minutesat 72° C. After the last cycle, a final extension of six minutes at 72°C. allowed ample time for full extension of products.

[0166] Samples were purified using a Wizard PCR Clean Up kit asdescribed, and double digested with NcoI/HindIII. These amplificationproducts were purified again using a Wizard PCR Clean Up kit. Inaddition, all six different linker length molecules for the 39/40breakpoint were cloned into NcoI/HindIII/SAP-treated pMON3977 as singleproteins (pMON32365, pMON32366, pMON32367, pMON32368, pMON32369 and32370). Subsequent steps leading to final DNA sequence confirmation weredone as described above.

EXAMPLE 7

[0167] Additional sequence rearranged Flt3 ligands were constructedusing the dimer template intermediates previously described. Forsequence rearranged Flt3 ligands having the fifteen amino acid linker(GlyGlyGlySer)₃GlyGlyGly SEQ ID NO:55, the dimer intermediates Flt4C.seqand Flt11N.seq were used as the template in the PCR reaction. Five newbreakpoints corresponding to Flt3 ligand amino acid residues 28/29,34/35, 62/63, 94/95, and 98/99, were constructed using a PCR basedapproach using a PCR Optimizer kit (Invitrogen) and the following primerpairs; FL29For/FL29Rev, FL35For/FL35Rev, FL63For/FL63Rev,FL95For/FL95Rev, FL99For/FL99Rev. Amplification conditions were asfollows: seven cycles of 94° C. for 1′, 62° C. for 2′, and 2.5′ at 70°C.; twelve cycles of 94° C. for 1′, 68° C. for 2′, and 70° C. for 2.5′;followed by a final cycle of 7′ at 72° C. PCR products corresponding tothe predicted insert size were digested to completion with NcoI andHindIII, and gel purified as described previously using Gene Clean II(Bio 101) following the manufacturers suggested protocol. Samples wereresuspended in 10 ul final volume with dH₂O. Inserts were cloned assingle genes into the mammalian expression vector pMON3977(NcoI/HindIII/SAP treated) and designated pMON35712, pMON35713,pMON35714, pMON35715, pMON35716, pMON35717, pMON35718 respectively.[0094] Additional techniques for the construction of the variant genes,recombinant protein expression, protein purification, proteincharacterization, biological activity determination can be found in WO94/12639, WO 94/12638, WO 95/20976, WO 95/21197, WO 95/20977, WO95/21254 and WO 96/23888 which are hereby incorporated by reference intheir entirety.

[0168] All references, patents or applications cited herein areincorporated by reference in their entirety as if written herein.

[0169] Various other examples will be apparent to the person skilled inthe art after reading the present disclosure without departing from thespirit and scope of the invention. It is intended that all such otherexamples be included within the scope of the appended claims.

1 151 135 amino acids amino acid single linear None 1 Ala Thr Gln AspCys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe 1 5 10 15 Ala Val LysIle Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 20 25 30 Val Thr ValAla Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Ala Leu 35 40 45 Trp Arg LeuVal Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val 50 55 60 Ala Gly SerLys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 65 70 75 80 His PheVal Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg 85 90 95 Phe ValGln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln 100 105 110 LeuVal Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys 115 120 125Leu Glu Leu Gln Cys Gln Pro 130 135 140 amino acids amino acid singlelinear None 2 Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser SerAsp Phe 1 5 10 15 Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu GlnAsp Tyr Pro 20 25 30 Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu CysGly Gly Leu 35 40 45 Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg LeuLys Thr Val 50 55 60 Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val AsnThr Glu Ile 65 70 75 80 His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro ProSer Cys Leu Arg 85 90 95 Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln GluThr Ser Glu Gln 100 105 110 Leu Val Ala Leu Lys Pro Trp Ile Thr Arg GlnAsn Phe Ser Arg Cys 115 120 125 Leu Glu Leu Gln Cys Gln Pro Asp Ser SerThr Leu 130 135 140 122 amino acids amino acid single linear None 3 AlaThr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe 1 5 10 15Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 20 25 30Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu 35 40 45Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val 50 55 60Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 65 70 7580 His Phe Val Thr Lys Cys Ala Phe Gln Glu Thr Ser Glu Gln Leu Val 85 9095 Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu 100105 110 Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu 115 120 135 amino acidsamino acid single linear None 4 Gly Thr Gln Asp Cys Ser Phe Gln His SerPro Ile Ser Ser Asp Phe 1 5 10 15 Ala Val Lys Ile Arg Glu Leu Ser AspTyr Leu Leu Gln Asp Tyr Pro 20 25 30 Val Thr Val Ala Ser Asn Leu Gln AspGlu Glu Leu Cys Gly Gly Leu 35 40 45 Trp Arg Leu Val Leu Ala Gln Arg TrpMet Glu Arg Leu Lys Thr Val 50 55 60 Ala Gly Ser Lys Met Gln Gly Leu LeuGlu Arg Val Asn Thr Glu Ile 65 70 75 80 His Phe Val Thr Lys Cys Ala PheGln Pro Pro Pro Ser Cys Leu Arg 85 90 95 Phe Val Gln Thr Asn Ile Ser ArgLeu Leu Gln Glu Thr Ser Glu Gln 100 105 110 Leu Val Ala Leu Lys Pro TrpIle Thr Arg Gln Asn Phe Ser Arg Cys 115 120 125 Leu Glu Leu Gln Cys GlnPro 130 135 140 amino acids amino acid single linear None 5 Gly Thr GlnAsp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe 1 5 10 15 Ala ValLys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 20 25 30 Val ThrVal Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu 35 40 45 Trp ArgLeu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val 50 55 60 Ala GlySer Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 65 70 75 80 HisPhe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg 85 90 95 PheVal Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln 100 105 110Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys 115 120125 Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu 130 135 140 135amino acids amino acid single linear None 6 Ala Thr Gln Asp Cys Ser PheGln His Ser Pro Ile Ser Ser Asp Phe 1 5 10 15 Ala Val Lys Ile Arg GluLeu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 20 25 30 Val Thr Val Ala Ser AsnLeu Gln Asp Glu Glu Leu Cys Gly Gly Leu 35 40 45 Trp Arg Leu Val Leu AlaGln Arg Trp Met Glu Arg Leu Lys Thr Val 50 55 60 Ala Gly Ser Lys Met GlnGly Leu Leu Glu Arg Val Asn Thr Glu Ile 65 70 75 80 His Phe Val Thr LysCys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg 85 90 95 Phe Val Gln Thr AsnIle Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln 100 105 110 Leu Val Ala LeuLys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys 115 120 125 Leu Glu LeuGln Cys Gln Pro 130 135 140 amino acids amino acid single linear None 7Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe 1 5 1015 Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 20 2530 Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu 35 4045 Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val 50 5560 Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 65 7075 80 His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg 8590 95 Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln100 105 110 Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser ArgCys 115 120 125 Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu 130 135140 155 amino acids amino acid single linear None 8 Ala Asp Glu Glu LeuCys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 1 5 10 15 Arg Trp Met GluArg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly 20 25 30 Leu Leu Glu ArgVal Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala 35 40 45 Phe Gln Pro ProPro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 50 55 60 Arg Leu Leu GlnGlu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 65 70 75 80 Ile Thr ArgGln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 85 90 95 Asp Ser SerThr Leu Ser Gly Gly Asn Gly Ser Gly Gly Asn Gly Ser 100 105 110 Gly GlyAsn Gly Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser 115 120 125 SerAsp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln 130 135 140Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln 145 150 155 150 amino acidsamino acid single linear None 9 Ala Asp Glu Glu Leu Cys Gly Gly Leu TrpArg Leu Val Leu Ala Gln 1 5 10 15 Arg Trp Met Glu Arg Leu Lys Thr ValAla Gly Ser Lys Met Gln Gly 20 25 30 Leu Leu Glu Arg Val Asn Thr Glu IleHis Phe Val Thr Lys Cys Ala 35 40 45 Phe Gln Pro Pro Pro Ser Cys Leu ArgPhe Val Gln Thr Asn Ile Ser 50 55 60 Arg Leu Leu Gln Glu Thr Ser Glu GlnLeu Val Ala Leu Lys Pro Trp 65 70 75 80 Ile Thr Arg Gln Asn Phe Ser ArgCys Leu Glu Leu Gln Cys Gln Pro 85 90 95 Asp Ser Ser Thr Leu Ser Gly GlyAsn Gly Ser Gly Gly Asn Gly Thr 100 105 110 Gln Asp Cys Ser Phe Gln HisSer Pro Ile Ser Ser Asp Phe Ala Val 115 120 125 Lys Ile Arg Glu Leu SerAsp Tyr Leu Leu Gln Asp Tyr Pro Val Thr 130 135 140 Val Ala Ser Asn LeuGln 145 150 145 amino acids amino acid single linear None 10 Ala Asp GluGlu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 1 5 10 15 Arg TrpMet Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly 20 25 30 Leu LeuGlu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala 35 40 45 Phe GlnPro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 50 55 60 Arg LeuLeu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 65 70 75 80 IleThr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 85 90 95 AspSer Ser Thr Leu Ser Gly Gly Asn Gly Thr Gln Asp Cys Ser Phe 100 105 110Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu 115 120125 Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu 130135 140 Gln 145 155 amino acids amino acid single linear None 11 Ala SerLys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His 1 5 10 15 PheVal Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 20 25 30 ValGln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 35 40 45 ValAla Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 50 55 60 GluLeu Gln Cys Gln Pro Asp Ser Ser Thr Leu Ser Gly Gly Asn Gly 65 70 75 80Ser Gly Gly Asn Gly Ser Gly Gly Asn Gly Thr Gln Asp Cys Ser Phe 85 90 95Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu 100 105110 Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu 115120 125 Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln130 135 140 Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly 145 150 155 150amino acids amino acid single linear None 12 Ala Ser Lys Met Gln Gly LeuLeu Glu Arg Val Asn Thr Glu Ile His 1 5 10 15 Phe Val Thr Lys Cys AlaPhe Gln Pro Pro Pro Ser Cys Leu Arg Phe 20 25 30 Val Gln Thr Asn Ile SerArg Leu Leu Gln Glu Thr Ser Glu Gln Leu 35 40 45 Val Ala Leu Lys Pro TrpIle Thr Arg Gln Asn Phe Ser Arg Cys Leu 50 55 60 Glu Leu Gln Cys Gln ProAsp Ser Ser Thr Leu Ser Gly Gly Asn Gly 65 70 75 80 Ser Gly Gly Asn GlyThr Gln Asp Cys Ser Phe Gln His Ser Pro Ile 85 90 95 Ser Ser Asp Phe AlaVal Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu 100 105 110 Gln Asp Tyr ProVal Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu 115 120 125 Cys Gly GlyLeu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg 130 135 140 Leu LysThr Val Ala Gly 145 150 145 amino acids amino acid single linear None 13Ala Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His 1 5 1015 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 20 2530 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 35 4045 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 50 5560 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Ser Gly Gly Asn Gly 65 7075 80 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala 8590 95 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val100 105 110 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly LeuTrp 115 120 125 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 130 135 140 Gly 145 155 amino acids amino acid single linearNone 14 Ala Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu1 5 10 15 Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp IleThr 20 25 30 Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro AspSer 35 40 45 Ser Thr Leu Ser Gly Gly Asn Gly Ser Gly Gly Asn Gly Ser GlyGly 50 55 60 Asn Gly Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser SerAsp 65 70 75 80 Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu GlnAsp Tyr 85 90 95 Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu CysGly Gly 100 105 110 Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu ArgLeu Lys Thr 115 120 125 Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu ArgVal Asn Thr Glu 130 135 140 Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro145 150 155 150 amino acids amino acid single linear None 15 Ala Pro ProSer Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu 1 5 10 15 Leu GlnGlu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr 20 25 30 Arg GlnAsn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser 35 40 45 Ser ThrLeu Ser Gly Gly Asn Gly Ser Gly Gly Asn Gly Thr Gln Asp 50 55 60 Cys SerPhe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile 65 70 75 80 ArgGlu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala 85 90 95 SerAsn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val 100 105 110Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys 115 120125 Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr 130135 140 Lys Cys Ala Phe Gln Pro 145 150 145 amino acids amino acidsingle linear None 16 Ala Pro Pro Ser Cys Leu Arg Phe Val Gln Thr AsnIle Ser Arg Leu 1 5 10 15 Leu Gln Glu Thr Ser Glu Gln Leu Val Ala LeuLys Pro Trp Ile Thr 20 25 30 Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu GlnCys Gln Pro Asp Ser 35 40 45 Ser Thr Leu Ser Gly Gly Asn Gly Thr Gln AspCys Ser Phe Gln His 50 55 60 Ser Pro Ile Ser Ser Asp Phe Ala Val Lys IleArg Glu Leu Ser Asp 65 70 75 80 Tyr Leu Leu Gln Asp Tyr Pro Val Thr ValAla Ser Asn Leu Gln Asp 85 90 95 Glu Glu Leu Cys Gly Gly Leu Trp Arg LeuVal Leu Ala Gln Arg Trp 100 105 110 Met Glu Arg Leu Lys Thr Val Ala GlySer Lys Met Gln Gly Leu Leu 115 120 125 Glu Arg Val Asn Thr Glu Ile HisPhe Val Thr Lys Cys Ala Phe Gln 130 135 140 Pro 145 155 amino acidsamino acid single linear None 17 Ala Asp Glu Glu Leu Cys Gly Gly Leu TrpArg Leu Val Leu Ala Gln 1 5 10 15 Arg Trp Met Glu Arg Leu Lys Thr ValAla Gly Ser Lys Met Gln Gly 20 25 30 Leu Leu Glu Arg Val Asn Thr Glu IleHis Phe Val Thr Lys Cys Ala 35 40 45 Phe Gln Pro Pro Pro Ser Cys Leu ArgPhe Val Gln Thr Asn Ile Ser 50 55 60 Arg Leu Leu Gln Glu Thr Ser Glu GlnLeu Val Ala Leu Lys Pro Trp 65 70 75 80 Ile Thr Arg Gln Asn Phe Ser ArgCys Leu Glu Leu Gln Cys Gln Pro 85 90 95 Asp Ser Ser Thr Leu Ser Gly GlySer Gly Ser Gly Gly Ser Gly Ser 100 105 110 Gly Gly Ser Gly Thr Gln AspCys Ser Phe Gln His Ser Pro Ile Ser 115 120 125 Ser Asp Phe Ala Val LysIle Arg Glu Leu Ser Asp Tyr Leu Leu Gln 130 135 140 Asp Tyr Pro Val ThrVal Ala Ser Asn Leu Gln 145 150 155 150 amino acids amino acid singlelinear None 18 Ala Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val LeuAla Gln 1 5 10 15 Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser LysMet Gln Gly 20 25 30 Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val ThrLys Cys Ala 35 40 45 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln ThrAsn Ile Ser 50 55 60 Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala LeuLys Pro Trp 65 70 75 80 Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu LeuGln Cys Gln Pro 85 90 95 Asp Ser Ser Thr Leu Ser Gly Gly Ser Gly Ser GlyGly Ser Gly Thr 100 105 110 Gln Asp Cys Ser Phe Gln His Ser Pro Ile SerSer Asp Phe Ala Val 115 120 125 Lys Ile Arg Glu Leu Ser Asp Tyr Leu LeuGln Asp Tyr Pro Val Thr 130 135 140 Val Ala Ser Asn Leu Gln 145 150 145amino acids amino acid single linear None 19 Met Ala Thr Gln Asp Cys SerPhe Gln His Ser Pro Ile Ser Ser Asp 1 5 10 15 Phe Ala Val Lys Ile ArgGlu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr 20 25 30 Pro Val Thr Val Ala SerAsn Leu Gln Asp Glu Glu Leu Cys Gly Gly 35 40 45 Leu Trp Arg Leu Val LeuAla Gln Arg Trp Met Glu Arg Leu Lys Thr 50 55 60 Val Ala Gly Ser Lys MetGln Gly Leu Leu Glu Arg Val Asn Thr Glu 65 70 75 80 Ile His Phe Val ThrLys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu 85 90 95 Arg Phe Val Gln ThrAsn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu 100 105 110 Gln Leu Val AlaLeu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg 115 120 125 Cys Leu GluLeu Gln Cys Gln Pro Asp Ser Ser Thr Leu Gly Gly Gly 130 135 140 Ser 145143 amino acids amino acid single linear None 20 Gly Ser Gly Gly Thr GlnAsp Cys Ser Phe Gln His Ser Pro Ile Ser 1 5 10 15 Ser Asp Phe Ala ValLys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln 20 25 30 Asp Tyr Pro Val ThrVal Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys 35 40 45 Gly Gly Leu Trp ArgLeu Val Leu Ala Gln Arg Trp Met Glu Arg Leu 50 55 60 Lys Thr Val Ala GlySer Lys Met Gln Gly Leu Leu Glu Arg Val Asn 65 70 75 80 Thr Glu Ile HisPhe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser 85 90 95 Cys Leu Arg PheVal Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr 100 105 110 Ser Glu GlnLeu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe 115 120 125 Ser ArgCys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu 130 135 140 149amino acids amino acid single linear None 21 Met Ala Thr Gln Asp Cys SerPhe Gln His Ser Pro Ile Ser Ser Asp 1 5 10 15 Phe Ala Val Lys Ile ArgGlu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr 20 25 30 Pro Val Thr Val Ala SerAsn Leu Gln Asp Glu Glu Leu Cys Gly Gly 35 40 45 Leu Trp Arg Leu Val LeuAla Gln Arg Trp Met Glu Arg Leu Lys Thr 50 55 60 Val Ala Gly Ser Lys MetGln Gly Leu Leu Glu Arg Val Asn Thr Glu 65 70 75 80 Ile His Phe Val ThrLys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu 85 90 95 Arg Phe Val Gln ThrAsn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu 100 105 110 Gln Leu Val AlaLeu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg 115 120 125 Cys Leu GluLeu Gln Cys Gln Pro Asp Ser Ser Thr Leu Gly Gly Gly 130 135 140 Ser GlyGly Gly Ser 145 144 amino acids amino acid single linear None 22 Gly SerGly Gly Gly Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile 1 5 10 15 SerSer Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu 20 25 30 GlnAsp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu 35 40 45 CysGly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg 50 55 60 LeuLys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val 65 70 75 80Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro 85 90 95Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu 100 105110 Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn 115120 125 Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu130 135 140 153 amino acids amino acid single linear None 23 Met Ala ThrGln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp 1 5 10 15 Phe AlaVal Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr 20 25 30 Pro ValThr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly 35 40 45 Leu TrpArg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr 50 55 60 Val AlaGly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu 65 70 75 80 IleHis Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu 85 90 95 ArgPhe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu 100 105 110Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg 115 120125 Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Gly Gly Gly 130135 140 Ser Gly Gly Gly Ser Gly Gly Gly Ser 145 150 150 amino acidsamino acid single linear None 24 Gly Ser Gly Gly Gly Ser Gly Gly Gly SerGly Thr Gln Asp Cys Ser 1 5 10 15 Phe Gln His Ser Pro Ile Ser Ser AspPhe Ala Val Lys Ile Arg Glu 20 25 30 Leu Ser Asp Tyr Leu Leu Gln Asp TyrPro Val Thr Val Ala Ser Asn 35 40 45 Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp Arg Leu Val Leu Ala 50 55 60 Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala Gly Ser Lys Met Gln 65 70 75 80 Gly Leu Leu Glu Arg Val Asn ThrGlu Ile His Phe Val Thr Lys Cys 85 90 95 Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe Val Gln Thr Asn Ile 100 105 110 Ser Arg Leu Leu Gln Glu ThrSer Glu Gln Leu Val Ala Leu Lys Pro 115 120 125 Trp Ile Thr Arg Gln AsnPhe Ser Arg Cys Leu Glu Leu Gln Cys Gln 130 135 140 Pro Asp Ser Ser ThrLeu 145 150 146 amino acids amino acid single linear None 25 Ala Asp GluGlu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 1 5 10 15 Arg TrpMet Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly 20 25 30 Leu LeuGlu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala 35 40 45 Phe GlnPro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 50 55 60 Arg LeuLeu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 65 70 75 80 IleThr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 85 90 95 AspSer Ser Thr Leu Gly Gly Gly Ser Gly Gly Thr Gln Asp Cys Ser 100 105 110Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu 115 120125 Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn 130135 140 Leu Gln 145 147 amino acids amino acid single linear None 26 AlaAsp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 1 5 10 15Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly 20 25 30Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala 35 40 45Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 50 55 60Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 65 70 7580 Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 85 9095 Asp Ser Ser Thr Leu Gly Gly Gly Ser Gly Gly Gly Thr Gln Asp Cys 100105 110 Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg115 120 125 Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val AlaSer 130 135 140 Asn Leu Gln 145 150 amino acids amino acid single linearNone 27 Ala Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln1 5 10 15 Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met GlnGly 20 25 30 Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys CysAla 35 40 45 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn IleSer 50 55 60 Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys ProTrp 65 70 75 80 Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln CysGln Pro 85 90 95 Asp Ser Ser Thr Leu Gly Gly Gly Ser Gly Gly Gly Ser GlyGly Thr 100 105 110 Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser AspPhe Ala Val 115 120 125 Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln AspTyr Pro Val Thr 130 135 140 Val Ala Ser Asn Leu Gln 145 150 153 aminoacids amino acid single linear None 28 Ala Asp Glu Glu Leu Cys Gly GlyLeu Trp Arg Leu Val Leu Ala Gln 1 5 10 15 Arg Trp Met Glu Arg Leu LysThr Val Ala Gly Ser Lys Met Gln Gly 20 25 30 Leu Leu Glu Arg Val Asn ThrGlu Ile His Phe Val Thr Lys Cys Ala 35 40 45 Phe Gln Pro Pro Pro Ser CysLeu Arg Phe Val Gln Thr Asn Ile Ser 50 55 60 Arg Leu Leu Gln Glu Thr SerGlu Gln Leu Val Ala Leu Lys Pro Trp 65 70 75 80 Ile Thr Arg Gln Asn PheSer Arg Cys Leu Glu Leu Gln Cys Gln Pro 85 90 95 Asp Ser Ser Thr Leu GlyGly Gly Ser Gly Gly Gly Ser Gly Gly Gly 100 105 110 Ser Gly Thr Gln AspCys Ser Phe Gln His Ser Pro Ile Ser Ser Asp 115 120 125 Phe Ala Val LysIle Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr 130 135 140 Pro Val ThrVal Ala Ser Asn Leu Gln 145 150 155 amino acids amino acid single linearNone 29 Ala Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln1 5 10 15 Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met GlnGly 20 25 30 Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys CysAla 35 40 45 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn IleSer 50 55 60 Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys ProTrp 65 70 75 80 Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln CysGln Pro 85 90 95 Asp Ser Ser Thr Leu Gly Gly Gly Ser Gly Gly Gly Ser GlyGly Gly 100 105 110 Ser Gly Gly Gly Thr Gln Asp Cys Ser Phe Gln His SerPro Ile Ser 115 120 125 Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser AspTyr Leu Leu Gln 130 135 140 Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln145 150 155 161 amino acids amino acid single linear None 30 Ala Asp GluGlu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln 1 5 10 15 Arg TrpMet Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly 20 25 30 Leu LeuGlu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala 35 40 45 Phe GlnPro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 50 55 60 Arg LeuLeu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 65 70 75 80 IleThr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 85 90 95 AspSer Ser Thr Leu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 100 105 110Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Thr Gln Asp Cys Ser Phe 115 120125 Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu 130135 140 Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu145 150 155 160 Gln 155 amino acids amino acid single linear None 31 AlaAsp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu 1 5 10 15Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg 20 25 30Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val 35 40 45Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro 50 55 60Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu 65 70 7580 Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn 85 9095 Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu 100105 110 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Thr115 120 125 Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe AlaVal 130 135 140 Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln 145 150 155155 amino acids amino acid single linear None 32 Ala Ala Ser Asn Leu GlnAsp Glu Glu Leu Cys Gly Gly Leu Trp Arg 1 5 10 15 Leu Val Leu Ala GlnArg Trp Met Glu Arg Leu Lys Thr Val Ala Gly 20 25 30 Ser Lys Met Gln GlyLeu Leu Glu Arg Val Asn Thr Glu Ile His Phe 35 40 45 Val Thr Lys Cys AlaPhe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val 50 55 60 Gln Thr Asn Ile SerArg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val 65 70 75 80 Ala Leu Lys ProTrp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu 85 90 95 Leu Gln Cys GlnPro Asp Ser Ser Thr Leu Gly Gly Gly Ser Gly Gly 100 105 110 Gly Ser GlyGly Gly Ser Gly Gly Gly Thr Gln Asp Cys Ser Phe Gln 115 120 125 His SerPro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser 130 135 140 AspTyr Leu Leu Gln Asp Tyr Pro Val Thr Val 145 150 155 155 amino acidsamino acid single linear None 33 Ala Val Ala Gly Ser Lys Met Gln Gly LeuLeu Glu Arg Val Asn Thr 1 5 10 15 Glu Ile His Phe Val Thr Lys Cys AlaPhe Gln Pro Pro Pro Ser Cys 20 25 30 Leu Arg Phe Val Gln Thr Asn Ile SerArg Leu Leu Gln Glu Thr Ser 35 40 45 Glu Gln Leu Val Ala Leu Lys Pro TrpIle Thr Arg Gln Asn Phe Ser 50 55 60 Arg Cys Leu Glu Leu Gln Cys Gln ProAsp Ser Ser Thr Leu Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Ser Gly GlyGly Ser Gly Gly Gly Thr Gln Asp 85 90 95 Cys Ser Phe Gln His Ser Pro IleSer Ser Asp Phe Ala Val Lys Ile 100 105 110 Arg Glu Leu Ser Asp Tyr LeuLeu Gln Asp Tyr Pro Val Thr Val Ala 115 120 125 Ser Asn Leu Gln Asp GluGlu Leu Cys Gly Gly Leu Trp Arg Leu Val 130 135 140 Leu Ala Gln Arg TrpMet Glu Arg Leu Lys Thr 145 150 155 155 amino acids amino acid singlelinear None 34 Ala Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 1 5 10 15 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 20 25 30 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 35 40 45 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe SerArg Cys Leu 50 55 60 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Gly GlyGly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Thr GlnAsp Cys Ser Phe 85 90 95 Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val LysIle Arg Glu Leu 100 105 110 Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val ThrVal Ala Ser Asn Leu 115 120 125 Gln Asp Glu Glu Leu Cys Gly Gly Leu TrpArg Leu Val Leu Ala Gln 130 135 140 Arg Trp Met Glu Arg Leu Lys Thr ValAla Gly 145 150 155 155 amino acids amino acid single linear None 35 AlaPro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu 1 5 10 15Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr 20 25 30Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser 35 40 45Ser Thr Leu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 50 55 60Gly Gly Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp 65 70 7580 Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr 85 9095 Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly 100105 110 Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr115 120 125 Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn ThrGlu 130 135 140 Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro 145 150 155155 amino acids amino acid single linear None 36 Ala Arg Phe Val Gln ThrAsn Ile Ser Arg Leu Leu Gln Glu Thr Ser 1 5 10 15 Glu Gln Leu Val AlaLeu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser 20 25 30 Arg Cys Leu Glu LeuGln Cys Gln Pro Asp Ser Ser Thr Leu Gly Gly 35 40 45 Gly Ser Gly Gly GlySer Gly Gly Gly Ser Gly Gly Gly Thr Gln Asp 50 55 60 Cys Ser Phe Gln HisSer Pro Ile Ser Ser Asp Phe Ala Val Lys Ile 65 70 75 80 Arg Glu Leu SerAsp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala 85 90 95 Ser Asn Leu GlnAsp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val 100 105 110 Leu Ala GlnArg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys 115 120 125 Met GlnGly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr 130 135 140 LysCys Ala Phe Gln Pro Pro Pro Ser Cys Leu 145 150 155 155 amino acidsamino acid single linear None 37 Ala Thr Asn Ile Ser Arg Leu Leu Gln GluThr Ser Glu Gln Leu Val 1 5 10 15 Ala Leu Lys Pro Trp Ile Thr Arg GlnAsn Phe Ser Arg Cys Leu Glu 20 25 30 Leu Gln Cys Gln Pro Asp Ser Ser ThrLeu Gly Gly Gly Ser Gly Gly 35 40 45 Gly Ser Gly Gly Gly Ser Gly Gly GlyThr Gln Asp Cys Ser Phe Gln 50 55 60 His Ser Pro Ile Ser Ser Asp Phe AlaVal Lys Ile Arg Glu Leu Ser 65 70 75 80 Asp Tyr Leu Leu Gln Asp Tyr ProVal Thr Val Ala Ser Asn Leu Gln 85 90 95 Asp Glu Glu Leu Cys Gly Gly LeuTrp Arg Leu Val Leu Ala Gln Arg 100 105 110 Trp Met Glu Arg Leu Lys ThrVal Ala Gly Ser Lys Met Gln Gly Leu 115 120 125 Leu Glu Arg Val Asn ThrGlu Ile His Phe Val Thr Lys Cys Ala Phe 130 135 140 Gln Pro Pro Pro SerCys Leu Arg Phe Val Gln 145 150 155 4 amino acids amino acid singlelinear None 38 Gly Gly Gly Ser 1 8 amino acids amino acid single linearNone 39 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 12 amino acids amino acidsingle linear None 40 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 15 10 7 amino acids amino acid single linear None 41 Ser Gly Gly Ser GlyGly Ser 1 5 5 amino acids amino acid single linear None 42 Glu Phe GlyAsn Met 1 5 6 amino acids amino acid single linear None 43 Glu Phe GlyGly Asn Met 1 5 9 amino acids amino acid single linear None 44 Glu PheGly Gly Asn Gly Gly Asn Met 1 5 7 amino acids amino acid single linearNone 45 Gly Gly Ser Asp Met Ala Gly 1 5 5 amino acids amino acid singlelinear None 46 Ser Gly Gly Asn Gly 1 5 10 amino acids amino acid singlelinear None 47 Ser Gly Gly Asn Gly Ser Gly Gly Asn Gly 1 5 10 15 aminoacids amino acid single linear None 48 Ser Gly Gly Asn Gly Ser Gly GlyAsn Gly Ser Gly Gly Asn Gly 1 5 10 15 10 amino acids amino acid singlelinear None 49 Ser Gly Gly Ser Gly Ser Gly Gly Ser Gly 1 5 10 15 aminoacids amino acid single linear None 50 Ser Gly Gly Ser Gly Ser Gly GlySer Gly Ser Gly Gly Ser Gly 1 5 10 15 6 amino acids amino acid singlelinear None 51 Gly Gly Gly Ser Gly Gly 1 5 7 amino acids amino acidsingle linear None 52 Gly Gly Gly Ser Gly Gly Gly 1 5 10 amino acidsamino acid single linear None 53 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly1 5 10 13 amino acids amino acid single linear None 54 Gly Gly Gly SerGly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 15 amino acids amino acidsingle linear None 55 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly SerGly Gly Gly 1 5 10 15 21 amino acids amino acid single linear None 56Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 1015 Gly Gly Gly Ser Gly 20 33 base pairs nucleic acid single linear 57CTGACCATGG CNACCCAGGA CTGCTCCTTC CAA 33 32 base pairs nucleic acidsingle linear 58 ACTGAAGCTT AGGGCTGACA CTGCAGCTCC AG 32 32 base pairsnucleic acid single linear 59 ACTGAAGCTT ACAGGGTTGA GGAGTCGGGC TG 32 46base pairs nucleic acid single linear 60 GACTGCCATG GCNACYCAGGAYTGYTCYTT YCAACACAGC CCCATC 46 46 base pairs nucleic acid single linear61 GACTGCCATG GCNACYCAGG AYTGYTCYTT YCAACACAGC CCCATC 46 22 base pairsnucleic acid single linear 62 TGTCCAAACT CATCAATGTA TC 22 38 base pairsnucleic acid single linear 63 CATGGCCATG GCCGACGAGG AGCTCTGCGG GGGCCTCT38 36 base pairs nucleic acid single linear 64 GCTAGAAGCT TACTGCAGGTTGGAGGCCAC GGTGAC 36 38 base pairs nucleic acid single linear 65CATGGCCATG GCCTCCAAGA TGCAAGGCTT GCTGGAGC 38 36 base pairs nucleic acidsingle linear 66 GCTAGAAGCT TACCCAGCGA CAGTCTTGAG CCGCTC 36 36 basepairs nucleic acid single linear 67 CATGGCCATG GCCCCCCCCA GCTGTCTTCGCTTCGT 36 37 base pairs nucleic acid single linear 68 GCTAGAAGCTTAGGGCTGAA AGGCACATTT GGTGACA 37 42 base pairs nucleic acid singlelinear 69 CCCTGTCTGG CGGCAACGGC ACCCAGGACT GCTCCTTCCA AC 42 48 basepairs nucleic acid single linear 70 GCGGTAACGG CAGTGGAGGT AATGGCACCCAGGACTGCTC CTTCCAAC 48 57 base pairs nucleic acid single linear 71ACGGCAGTGG TGGCAATGGG AGCGGCGGAA ATGGAACCCA GGACTGCTCC TTCCAAC 57 38base pairs nucleic acid single linear 72 GTGCCGTTGC CGCCAGACAGGGTTGAGGAG TCGGGCTG 38 48 base pairs nucleic acid single linear 73ATTACCTCCA CTGCCGTTAC CGCCTGACAG GGTTGAGGAG TCGGGCTG 48 54 base pairsnucleic acid single linear 74 GCTCCCATTG CCACCACTGC CGTTACCTCCAGACAGGGTT GAGGAGTCGG GCTG 54 60 base pairs nucleic acid single linear75 GATGAGGATC CGGTGGCAAT GGGAGCGGCG GAAATGGAAC CCAGGACTGC TCCTTCCAC 6045 base pairs nucleic acid single linear 76 GATGACGGAT CCGTTACCTCCAGACAGGGT TGAGGAGTCG GGCTG 45 46 base pairs nucleic acid single linear77 GATGACGGAT CCGGAGGTAA TGGCACCCAG GACTGCTCCT TCCAAC 46 29 base pairsnucleic acid single linear 78 GACTGCCATG GCCGACGAGG AGCTCTGCG 29 28 basepairs nucleic acid single linear 79 GACTCAAGCT TACTGCAGGT TGGAGGCC 28 39base pairs nucleic acid single linear 80 GACTCGGGAT CCGGAGGTTCTGGCACCCAG GACTGCTCC 39 41 base pairs nucleic acid single linear 81GACTGGGATC CGGTGGCAGT GGGAGCGGCG GATCTGGAAC C 41 39 base pairs nucleicacid single linear 82 GACTTGGGAT CCACTACCTC CAGACAGGGT TGAGGAGTC 39 39base pairs nucleic acid single linear 83 ACTGACGGAT CCACCGCCCAGGGTTGAGGA GTCGGGCTG 39 51 base pairs nucleic acid single linear 84ACTGACGGAT CCACCTCCTG ACCCACCGCC CAGGGTTGAG GAGTCGGGCT G 51 63 basepairs nucleic acid single linear 85 ACTGACGGAT CCACCTCCTG ACCCACCTCCTGACCCACCG CCCAGGGTTG AGGAGTCGG60 CTG 63 28 base pairs nucleic acidsingle linear 86 ACGTAAAGCT TACAGGGTTG AGGAGTCG 28 40 base pairs nucleicacid single linear 87 GTCAGTGGAT CCGGAGGTAC CCAGGACTGC TCCTTCCAAC 40 43base pairs nucleic acid single linear 88 GTCAGTGGAT CCGGAGGTGGCACCCAGGAC TGCTCCTTCC AAC 43 60 base pairs nucleic acid single linear 89GTCAGTGGAT CCGGAGGTGG CTCAGGGGGA GGTAGTGGTA CCCAGGACTG CTCCTTCCA 60 57base pairs nucleic acid single linear 90 GTTGCCATGG CNTCNAAYCTGCARGAYGAR GARCTGTGCG GGGGCCTCTG GCGGCTG 57 57 base pairs nucleic acidsingle linear 91 GTTGCCATGG CNAAYCTGCA RGAYGARGAR CTGTGYGGGG GCCTCTGGCGGCTGGTC 57 57 base pairs nucleic acid single linear 92 GTTGCCATGGCNCTGCARGA YGARGARCTG TGYGGYGGCC TCTGGCGGCT GGTCCTG 57 57 base pairsnucleic acid single linear 93 GTTGCCATGG CNCARGAYGA RGARCTGTGYGGYGGYCTCT GGCGGCTGGT CCTGGCA 57 57 base pairs nucleic acid singlelinear 94 GTTGCCATGG CNGAYGARGA RCTGTGYGGY GGYCTCTGGC GGCTGGTCCT GGCACAG57 57 base pairs nucleic acid single linear 95 GTTGCCATGG CNGARGARCTGTGYGGYGGY CTCTGGCGGC TGGTCCTGGC ACAGCGC 57 57 base pairs nucleic acidsingle linear 96 GTTGCCATGG CNGARCTGTG YGGYGGYCTG TGGCGYCTGG TCCTGGCACAGCGCTGG 57 57 base pairs nucleic acid single linear 97 GTTGCCATGGCNCTGTGYGG YGGYCTGTGG CGYCTGGTCC TGGCACAGCG CTGGATG 57 30 base pairsnucleic acid single linear 98 TATGCAAGCT TAGGCCACGG TGACTGGGTA 30 30base pairs nucleic acid single linear 99 TATGCAAGCT TAGGAGGCCACGGTGACTGG 30 30 base pairs nucleic acid single linear 100 TATGCAAGCTTAGTTGGAGG CCACGGTGAC 30 30 base pairs nucleic acid single linear 101TATGCAAGCT TACAGGTTGG AGGCCACGGT 30 30 base pairs nucleic acid singlelinear 102 TATGCAAGCT TACTGCAGGT TGGAGGCCAC 30 30 base pairs nucleicacid single linear 103 TATGCAAGCT TAGTCCTGCA GGTTGGAGGC 30 30 base pairsnucleic acid single linear 104 TATGCAAGCT TACTCGTCCT GCAGGTTGGA 30 30base pairs nucleic acid single linear 105 TATGCAAGCT TACTCCTCGTCCTGCAGGTT 30 405 base pairs nucleic acid single linear 106 GCCACCCAGGACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAAT 60 CGTGAGCTGTCTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGC 120 GACGAGGAGCTCTGCGGGGC GCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGC 180 CTCAAGACTGTCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGA 240 CACTTTGTCACCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGA 300 AACATCTCCCGCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGA 360 ACTCGCCAGAACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCC 405 420 base pairs nucleic acidsingle linear 107 GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGCTGTCAAAAT 60 CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTCCAACCTGC 120 GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTGGATGGAGC 180 CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAACACGGAGA 240 CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTTCGTCCAGA 300 AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAAGCCCTGGA 360 ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTCCTCAACCC 420 366 base pairs nucleic acid single linear 108 GCCACCCAGGACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAAT 60 CGTGAGCTGTCTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGC 120 GACGAGGAGCTCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGC 180 CTCAAGACTGTCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGA 240 CACTTTGTCACCAAATGTGC CTTTCAGGAG ACCTCCGAGC AGCTGGTGGC GCTGAAGC 300 TGGATCACTCGCCAGAACTT CTCCCGGTGC CTGGAGCTGC AGTGTCAGCC CGACTCCT 360 ACCCTG 366 405base pairs nucleic acid single linear 109 GGAACTCAGG ATTGTTCTTTCCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAAT 60 CGTGAGCTGT CTGACTACCTGCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGC 120 GACGAGGAGC TCTGCGGGGGCCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGC 180 CTCAAGACTG TCGCTGGGTCCAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGA 240 CACTTTGTCA CCAAATGTGCCTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGA 300 AACATCTCCC GCCTCCTGCAGGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGA 360 ACTCGCCAGA ACTTCTCCCGGTGCCTGGAG CTGCAGTGTC AGCCC 405 420 base pairs nucleic acid singlelinear 110 GGTACCCAGG ATTGTTCTTT CCAACACAGC CCCATCTCCT CCGACTTCGCTGTCAAAAT 60 CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTCCAACCTGC 120 GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTGGATGGAGC 180 CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAACACGGAGA 240 CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTTCGTCCAGA 300 AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAAGCCCTGGA 360 ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTCCTCAACCC 420 405 base pairs nucleic acid single linear 111 GCCACTCAGGACTGTTCTTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAAT 60 CGTGAGCTGTCTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGC 120 GACGAGGAGCTCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGC 180 CTCAAGACTGTCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGA 240 CACTTTGTCACCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGA 300 AACATCTCCCGCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGA 360 ACTCGCCAGAACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCC 405 420 base pairs nucleic acidsingle linear 112 GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGCTGTCAAAAT 60 CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTCCAACCTGC 120 GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTGGATGGAGC 180 CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAACACGGAGA 240 CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTTCGTCCAGA 300 AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAAGCCCTGGA 360 ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTCCTCAACCC 420 465 base pairs nucleic acid single linear 113 GCCGACGAGGAGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGA 60 CGGCTCAAGACTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGG 120 ATACACTTTGTCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG CTTCGTCC 180 ACCAACATCTCCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCT GAAGCCCT 240 ATCACTCGCCAGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGA CTCCTCAA 300 CTGTCTGGAGGTAACGGATC CGGTGGCAAT GGGAGCGGCG GAAATGGAAC CCAGGACT 360 TCCTTCCAACACAGCCCCAT CTCCTCCGAC TTCGCTGTCA AAATCCGTGA GCTGTCTG 420 TACCTGCTTCAAGATTACCC AGTCACCGTG GCCTCCAACC TGCAG 465 450 base pairs nucleic acidsingle linear 114 GCCGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCGCTGGATGGA 60 CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGTGAACACGG 120 ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCGCTTCGTCC 180 ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCTGAAGCCCT 240 ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGACTCCTCAA 300 CTGTCAGGCG GTAACGGCAG TGGAGGTAAT GGCACCCAGG ACTGCTCCTTCCAACACA 360 CCCATCTCCT CCGACTTCGC TGTCAAAATC CGTGAGCTGT CTGACTACCTGCTTCAAG 420 TACCCAGTCA CCGTGGCCTC CAACCTGCAG 450 435 base pairs nucleicacid single linear 115 GCCGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCCTGGCACAGCG CTGGATGGA 60 CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGCTGGAGCGCGT GAACACGG 120 ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCAGCTGTCTTCG CTTCGTCC 180 ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGCTGGTGGCGCT GAAGCCCT 240 ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGTGTCAGCCCGA CTCCTCAA 300 CTGTCTGGCG GCAACGGCAC CCAGGACTGC TCCTTCCAACACAGCCCCAT CTCCTCCG 360 TTCGCTGTCA AAATCCGTGA GCTGTCTGAC TACCTGCTTCAAGATTACCC AGTCACCG 420 GCCTCCAACC TGCAG 435 465 base pairs nucleic acidsingle linear 116 GCCTCCAAGA TGCAAGGCTT GCTGGAGCGC GTGAACACGG AGATACACTTTGTCACCAA 60 TGTGCCTTTC AGCCCCCCCC CAGCTGTCTT CGCTTCGTCC AGACCAACATCTCCCGCC 120 CTGCAGGAGA CCTCCGAGCA GCTGGTGGCG CTGAAGCCCT GGATCACTCGCCAGAACT 180 TCCCGGTGCC TGGAGCTGCA GTGTCAGCCC GACTCCTCAA CCCTGTCTGGAGGTAACG 240 TCCGGTGGCA ATGGGAGCGG CGGAAATGGA ACCCAGGACT GCTCCTTCCAACACAGCC 300 ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCTTCAAGATT 360 CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCTCTGGCGGC 420 GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGG 465 450base pairs nucleic acid single linear 117 GCCTCCAAGA TGCAAGGCTTGCTGGAGCGC GTGAACACGG AGATACACTT TGTCACCAA 60 TGTGCCTTTC AGCCCCCCCCCAGCTGTCTT CGCTTCGTCC AGACCAACAT CTCCCGCC 120 CTGCAGGAGA CCTCCGAGCAGCTGGTGGCG CTGAAGCCCT GGATCACTCG CCAGAACT 180 TCCCGGTGCC TGGAGCTGCAGTGTCAGCCC GACTCCTCAA CCCTGTCTGG AGGTAACG 240 TCCGGAGGTA ATGGCACCCAGGACTGCTCC TTCCAACACA GCCCCATCTC CTCCGACT 300 GCTGTCAAAA TCCGTGAGCTGTCTGACTAC CTGCTTCAAG ATTACCCAGT CACCGTGG 360 TCCAACCTGC AGGACGAGGAGCTCTGCGGG GGCCTCTGGC GGCTGGTCCT GGCACAGC 420 TGGATGGAGC GGCTCAAGACTGTCGCTGGG 450 435 base pairs nucleic acid single linear 118 GCCTCCAAGATGCAAGGCTT GCTGGAGCGC GTGAACACGG AGATACACTT TGTCACCAA 60 TGTGCCTTTCAGCCCCCCCC CAGCTGTCTT CGCTTCGTCC AGACCAACAT CTCCCGCC 120 CTGCAGGAGACCTCCGAGCA GCTGGTGGCG CTGAAGCCCT GGATCACTCG CCAGAACT 180 TCCCGGTGCCTGGAGCTGCA GTGTCAGCCC GACTCCTCAA CCCTGTCTGG CGGCAACG 240 ACGCAGGACTGCTCCTTCCA ACACAGCCCC ATCTCCTCCG ACTTCGCTGT CAAAATCC 300 GAGCTGTCTGACTACCTGCT TCAAGATTAC CCAGTCACCG TGGCCTCCAA CCTGCAGG 360 GAGGAGCTCTGCGGGGGCCT CTGGCGGCTG GTCCTGGCAC AGCGCTGGAT GGAGCGGC 420 AAGACTGTCGCTGGG 435 465 base pairs nucleic acid single linear 119 GCCCCCCCCAGCTGTCTTCG CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGAC 60 TCCGAGCAGCTGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCC 120 GAGCTGCAGTGTCAGCCCGA CTCCTCAACC CTGTCTGGAG GTAACGGCAG TGGTGGCA 180 GGGAGCGGTGGAAATGGAAC CCAGGACTGC TCCTTCCAAC ACAGCCCCAT CTCCTCCG 240 TTCGCTGTCAAAATCCGTGA GCTGTCTGAC TACCTGCTTC AAGATTACCC AGTCACCG 300 GCCTCCAACCTGCAGGACGA GGAGCTCTGC GGGGGCCTCT GGCGGCTGGT CCTGGCAC 360 CGCTGGATGGAGCGGCTCAA GACTGTCGCT GGGTCCAAGA TGCAAGGCTT GCTGGAGC 420 GTGAACACGGAGATACACTT TGTCACCAAA TGTGCCTTTC AGCCC 465 450 base pairs nucleic acidsingle linear 120 GCCCCCCCCA GCTGTCTTCG CTTCGTCCAG ACCAACATCT CCCGCCTCCTGCAGGAGAC 60 TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTCCCGGTGCC 120 GAGCTGCAGT GTCAGCCCGA CTCCTCAACC CTGTCAGGCG GTAACGGCAGTGGAGGTA 180 GGCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGCTGTCAAAA 240 CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTCCAACCTGC 300 GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTGGATGGAGC 360 CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAACACGGAGA 420 CACTTTGTCA CCAAATGTGC CTTTCAGCCC 450 435 base pairs nucleicacid single linear 121 GCCCCCCCCA GCTGTCTTCG CTTCGTCCAG ACCAACATCTCCCGCCTCCT GCAGGAGAC 60 TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG ATCACTCGCCAGAACTTCTC CCGGTGCC 120 GAGCTGCAGT GTCAGCCCGA CTCCTCAACC CTGTCTGGCGGCAACGGCAC GCAGGACT 180 TCCTTCCAAC ACAGCCCCAT CTCCTCCGAC TTCGCTGTCAAAATCCGTGA GCTGTCTG 240 TACCTGCTTC AAGATTACCC AGTCACCGTG GCCTCCAACCTGCAGGACGA GGAGCTCT 300 GGGGGCCTCT GGCGGCTGGT CCTGGCACAG CGCTGGATGGAGCGGCTCAA GACTGTCG 360 GGGTCCAAGA TGCAAGGCTT GCTGGAGCGC GTGAACACGGAGATACACTT TGTCACCA 420 TGTGCCTTTC AGCCC 435 451 base pairs nucleic acidsingle linear 122 GCCGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCGCTGGATGGA 60 CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGTGAACACGG 120 ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCGCTTCGTCC 180 ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCTGAAGCCCT 240 ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGACTCCTCAA 300 CTGTCTGGAG GTAGTGGATC CGGAGGTTCT GGCAACCCAG GACTGCTCCTTCCAACAC 360 CCCCATCTCC TCCGACTTCG CTGTCAAAAT CCGTGAGCTG TCTGACTACCTGCTTCAA 420 TTACCCAGTC ACCGTGGCCT CCAACCTGCA G 451 465 base pairsnucleic acid single linear 123 GCCGACGAGG AGCTCTGCGG GGGCCTCTGGCGGCTGGTCC TGGCACAGCG CTGGATGGA 60 CGGCTCAAGA CTGTCGCTGG GTCCAAGATGCAAGGCTTGC TGGAGCGCGT GAACACGG 120 ATACACTTTG TCACCAAATG TGCCTTTCAGCCCCCCCCCA GCTGTCTTCG CTTCGTCC 180 ACCAACATCT CCCGCCTCCT GCAGGAGACCTCCGAGCAGC TGGTGGCGCT GAAGCCCT 240 ATCACTCGCC AGAACTTCTC CCGGTGCCTGGAGCTGCAGT GTCAGCCCGA CTCCTCAA 300 CTGTCTGGAG GTAGTGGATC CGGTGGCAGTGGGAGCGGCG GATCTGGAAC CCAGGACT 360 TCCTTCCAAC ACAGCCCCAT CTCCTCCGACTTCGCTGTCA AAATCCGTGA GCTGTCTG 420 TACCTGCTTC AAGATTACCC AGTCACCGTGGCCTCCAACC TGCAG 465 437 base pairs nucleic acid single linear 124CCATGGCCAC CCAGGACTGC TCCTTCCAAC ACAGCCCCAT CTCCTCCGAC TTCGCTGTC 60AAATCCGTGA GCTGTCTGAC TACCTGCTTC AAGATTACCC AGTCACCGTG GCCTCCAA 120TGCAGGACGA GGAGCTCTGC GGGGGCCTCT GGCGGCTGGT CCTGGCACAG CGCTGGAT 180AGCGGCTCAA GACTGTCGCT GGGTCCAAGA TGCAAGGCTT GCTGGAGCGC GTGAACAC 240AGATACACTT TGTCACCAAA TGTGCCTTTC AGCCCCCCCC CAGCTGTCTT CGCTTCGT 300AGACCAACAT CTCCCGCCTC CTGCAGGAGA CCTCCGAGCA GCTGGTGGCG CTGAAGCC 360GGATCACTCG CCAGAACTTC TCCCGGTGCC TGGAGCTGCA GTGTCAGCCC GACTCCTC 420CCCTGGGCGG TGGATCC 437 436 base pairs nucleic acid single linear 125GGATCCGGAG GTACCCAGGA CTGCTCCTTC CAACACAGCC CCATCTCCTC CGACTTCGC 60GTCAAAATCC GTGAGCTGTC TGACTACCTG CTTCAAGATT ACCCAGTCAC CGTGGCCT 120AACCTGCAGG ACGAGGAGCT CTGCGGGGGC CTCTGGCGGC TGGTCCTGGC ACAGCGCT 180ATGGAGCGGC TCAAGACTGT CGCTGGGTCC AAGATGCAAG GCTTGCTGGA GCGCGTGA 240ACGGAGATAC ACTTTGTCAC CAAATGTGCC TTTCAGCCCC CCCCCAGCTG TCTTCGCT 300GTCCAGACCA ACATCTCCCG CCTCCTGCAG GAGACCTCCG AGCAGCTGGT GGCGCTGA 360CCCTGGATCA CTCGCCAGAA CTTCTCCCGG TGCCTGGAGC TGCAGTGTCA GCCCGACT 420TCAACCCTGT AAGCTT 436 449 base pairs nucleic acid single linear 126CCATGGCCAC CCAGGACTGC TCCTTCCAAC ACAGCCCCAT CTCCTCCGAC TTCGCTGTC 60AAATCCGTGA GCTGTCTGAC TACCTGCTTC AAGATTACCC AGTCACCGTG GCCTCCAA 120TGCAGGACGA GGAGCTCTGC GGGGGCCTCT GGCGGCTGGT CCTGGCACAG CGCTGGAT 180AGCGGCTCAA GACTGTCGCT GGGTCCAAGA TGCAAGGCTT GCTGGAGCGC GTGAACAC 240AGATACACTT TGTCACCAAA TGTGCCTTTC AGCCCCCCCC CAGCTGTCTT CGCTTCGT 300AGACCAACAT CTCCCGCCTC CTGCAGGAGA CCTCCGAGCA GCTGGTGGCG CTGAAGCC 360GGATCACTCG CCAGAACTTC TCCCGGTGCC TGGAGCTGCA GTGTCAGCCC GACTCCTC 420CCCTGGGCGG TGGGTCAGGA GGTGGATCC 449 439 base pairs nucleic acid singlelinear 127 GGATCCGGAG GTGGCACCCA GGACTGCTCC TTCCAACACA GCCCCATCTCCTCCGACTT 60 GCTGTCAAAA TCCGTGAGCT GTCTGACTAC CTGCTTCAAG ATTACCCAGTCACCGTGG 120 TCCAACCTGC AGGACGAGGA GCTCTGCGGG GGCCTCTGGC GGCTGGTCCTGGCACAGC 180 TGGATGGAGC GGCTCAAGAC TGTCGCTGGG TCCAAGATGC AAGGCTTGCTGGAGCGCG 240 AACACGGAGA TACACTTTGT CACCAAATGT GCCTTTCAGC CCCCCCCCAGCTGTCTTC 300 TTCGTCCAGA CCAACATCTC CCGCCTCCTG CAGGAGACCT CCGAGCAGCTGGTGGCGC 360 AAGCCCTGGA TCACTCGCCA GAACTTCTCC CGGTGCCTGG AGCTGCAGTGTCAGCCCG 420 TCCTCAACCC TGTAAGCTT 439 461 base pairs nucleic acid singlelinear 128 CCATGGCCAC CCAGGACTGC TCCTTCCAAC ACAGCCCCAT CTCCTCCGACTTCGCTGTC 60 AAATCCGTGA GCTGTCTGAC TACCTGCTTC AAGATTACCC AGTCACCGTGGCCTCCAA 120 TGCAGGACGA GGAGCTCTGC GGGGGCCTCT GGCGGCTGGT CCTGGCACAGCGCTGGAT 180 AGCGGCTCAA GACTGTCGCT GGGTCCAAGA TGCAAGGCTT GCTGGAGCGCGTGAACAC 240 AGATACACTT TGTCACCAAA TGTGCCTTTC AGCCCCCCCC CAGCTGTCTTCGCTTCGT 300 AGACCAACAT CTCCCGCCTC CTGCAGGAGA CCTCCGAGCA GCTGGTGGCGCTGAAGCC 360 GGATCACTCG CCAGAACTTC TCCCGGTGCC TGGAGCTGCA GTGTCAGCCCGACTCCTC 420 CCCTGGGCGG TGGGTCAGGA GGTGGGTCAG GAGGTGGATC C 461 457 basepairs nucleic acid single linear 129 GGATCCGGAG GTGGCTCAGG GGGAGGTAGTGGTACCCAGG ACTGCTCCTT CCAACACAG 60 CCCATCTCCT CCGACTTCGC TGTCAAAATCCGTGAGCTGT CTGACTACCT GCTTCAAG 120 TACCCAGTCA CCGTGGCCTC CAACCTGCAGGACGAGGAGC TCTGCGGGGG CCTCTGGC 180 CTGGTCCTGG CACAGCGCTG GATGGAGCGGCTCAAGACTG TCGCTGGGTC CAAGATGC 240 GGCTTGCTGG AGCGCGTGAA CACGGAGATACACTTTGTCA CCAAATGTGC CTTTCAGC 300 CCCCCCAGCT GTCTTCGCTT CGTCCAGACCAACATCTCCC GCCTCCTGCA GGAGACCT 360 GAGCAGCTGG TGGCGCTGAA GCCCTGGATCACTCGCCAGA ACTTCTCCCG GTGCCTGG 420 CTGCAGTGTC AGCCCGACTC CTCAACCCTGTAAGCTT 457 438 base pairs nucleic acid single linear 130 GCCGACGAGGAGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGA 60 CGGCTCAAGACTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGG 120 ATACACTTTGTCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG CTTCGTCC 180 ACCAACATCTCCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCT GAAGCCCT 240 ATCACTCGCCAGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGA CTCCTCAA 300 CTGGGCGGTGGATCCGGAGG TACCCAGGAC TGCTCCTTCC AACACAGCCC CATCTCCT 360 GACTTCGCTGTCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCA 420 GTGGCCTCCAACCTGCAG 438 441 base pairs nucleic acid single linear 131 GCCGACGAGGAGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGA 60 CGGCTCAAGACTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGG 120 ATACACTTTGTCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG CTTCGTCC 180 ACCAACATCTCCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCT GAAGCCCT 240 ATCACTCGCCAGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGA CTCCTCAA 300 CTGGGCGGTGGATCCGGAGG TGGCACCCAG GACTGCTCCT TCCAACACAG CCCCATCT 360 TCCGACTTCGCTGTCAAAAT CCGTGAGCTG TCTGACTACC TGCTTCAAGA TTACCCAG 420 ACCGTGGCCTCCAACCTGCA G 441 450 base pairs nucleic acid single linear 132GCCGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGA 60CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGG 120ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG CTTCGTCC 180ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCT GAAGCCCT 240ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGA CTCCTCAA 300CTGGGCGGTG GGTCAGGAGG TGGATCCGGA GGTACCCAGG ACTGCTCCTT CCAACACA 360CCCATCTCCT CCGACTTCGC TGTCAAAATC CGTGAGCTGT CTGACTACCT GCTTCAAG 420TACCCAGTCA CCGTGGCCTC CAACCTGCAG 450 459 base pairs nucleic acid singlelinear 133 GCCGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCGCTGGATGGA 60 CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGTGAACACGG 120 ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCGCTTCGTCC 180 ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCTGAAGCCCT 240 ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGACTCCTCAA 300 CTGGGCGGTG GATCCGGAGG TGGCTCAGGG GGAGGTAGTG GTACCCAGGACTGCTCCT 360 CAACACAGCC CCATCTCCTC CGACTTCGCT GTCAAAATCC GTGAGCTGTCTGACTACC 420 CTTCAAGATT ACCCAGTCAC CGTGGCCTCC AACCTGCAG 459 465 basepairs nucleic acid single linear 134 GCCGACGAGG AGCTCTGCGG GGGCCTCTGGCGGCTGGTCC TGGCACAGCG CTGGATGGA 60 CGGCTCAAGA CTGTCGCTGG GTCCAAGATGCAAGGCTTGC TGGAGCGCGT GAACACGG 120 ATACACTTTG TCACCAAATG TGCCTTTCAGCCCCCCCCCA GCTGTCTTCG CTTCGTCC 180 ACCAACATCT CCCGCCTCCT GCAGGAGACCTCCGAGCAGC TGGTGGCGCT GAAGCCCT 240 ATCACTCGCC AGAACTTCTC CCGGTGCCTGGAGCTGCAGT GTCAGCCCGA CTCCTCAA 300 CTGGGCGGTG GGTCAGGAGG TGGGTCAGGAGGTGGATCCG GAGGTGGCAC CCAGGACT 360 TCCTTCCAAC ACAGCCCCAT CTCCTCCGACTTCGCTGTCA AAATCCGTGA GCTGTCTG 420 TACCTGCTTC AAGATTACCC AGTCACCGTGGCCTCCAACC TGCAG 465 483 base pairs nucleic acid single linear 135GCCGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGA 60CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGG 120ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGCCTTCG CTTCGTCC 180ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCT GAAGCCCT 240ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGA CTCCTCAA 300CTGGGCGGTG GGTCAGGAGG TGGGTCAGGA GGTGGATCCG GAGGTGGCTC AGGGGGAG 360AGTGGTACCC AGGACTGCTC CTTCCAACAC AGCCCCATCT CCTCCGACTT CGCTGTCA 420ATCCGTGAGC TGTCTGACTA CCTGCTTCAA GATTACCCAG TCACCGTGGC CTCCAACC 480 CAG483 465 base pairs nucleic acid single linear 136 GCCGATTACC CAGTCACCGTGGCCTCCAAC CTGCAGGACG AGGAGCTCTG CGGGGGCCT 60 TGGCGGCTGG TCCTGGCACAGCGCTGGATG GAGCGGCTCA AGACTGTCGC TGGGTCCA 120 ATGCAAGGCT TGCTGGAGCGCGTGAACACG GAGATACACT TTGTCACCAA ATGTGCCT 180 CAGCCCCCCC CCAGCTGTCTTCGCTTCGTC CAGACCAACA TCTCCCGCCT CCTGCAGG 240 ACCTCCGAGC AGCTGGTGGCGCTGAAGCCC TGGATCACTC GCCAGAACTT CTCCCGGT 300 CTGGAGCTGC AGTGTCAGCCCGACTCCTCA ACCCTGGGCG GTGGGTCAGG AGGTGGGT 360 GGAGGTGGAT CCGGAGGTGGCACCCAGGAC TGCTCCTTCC AACACAGCCC CATCTCCT 420 GACTTCGCTG TCAAAATCCGTGAGCTGTCT GACTACCTGC TTCAA 465 465 base pairs nucleic acid singlelinear 137 GCCGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGGCC TCTGGCGGCTGGTCCTGGC 60 CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGGCTTGCTGG 120 CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCCCCCCAGCT 180 CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGAGCAGCTGG 240 GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCTGCAGTGTC 300 CCCGACTCCT CAACCCTGGG CGGTGGGTCA GGAGGTGGGT CAGGAGGTGGATCCGGAG 360 GGCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGCTGTCAAAA 420 CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTG 465 465base pairs nucleic acid single linear 138 GCCGTCGCTG GGTCCAAGATGCAAGGCTTG CTGGAGCGCG TGAACACGGA GATACACTT 60 GTCACCAAAT GTGCCTTTCAGCCCCCCCCC AGCTGTCTTC GCTTCGTCCA GACCAACA 120 TCCCGCCTCC TGCAGGAGACCTCCGAGCAG CTGGTGGCGC TGAAGCCCTG GATCACTC 180 CAGAACTTCT CCCGGTGCCTGGAGCTGCAG TGTCAGCCCG ACTCCTCAAC CCTGGGCG 240 GGGTCAGGAG GTGGGTCAGGAGGTGGATCC GGAGGTGGCA CCCAGGACTG CTCCTTCC 300 CACAGCCCCA TCTCCTCCGACTTCGCTGTC AAAATCCGTG AGCTGTCTGA CTACCTGC 360 CAAGATTACC CAGTCACCGTGGCCTCCAAC CTGCAGGACG AGGAGCTCTG CGGGGGCC 420 TGGCGGCTGG TCCTGGCACAGCGCTGGATG GAGCGGCTCA AGACT 465 465 base pairs nucleic acid singlelinear 139 GCCTCCAAGA TGCAAGGCTT GCTGGAGCGC GTGAACACGG AGATACACTTTGTCACCAA 60 TGTGCCTTTC AGCCCCCCCC CAGCTGTCTT CGCTTCGTCC AGACCAACATCTCCCGCC 120 CTGCAGGAGA CCTCCGAGCA GCTGGTGGCG CTGAAGCCCT GGATCACTCGCCAGAACT 180 TCCCGGTGCC TGGAGCTGCA GTGTCAGCCC GACTCCTCAA CCCTGGGCGGTGGGTCAG 240 GGTGGGTCAG GAGGTGGATC CGGAGGTGGC ACCCAGGACT GCTCCTTCCAACACAGCC 300 ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCTTCAAGATT 360 CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCTCTGGCGGC 420 GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGG 465 465base pairs nucleic acid single linear 140 GCCCCCCCCA GCTGTCTTCGCTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGAC 60 TCCGAGCAGC TGGTGGCGCTGAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCC 120 GAGCTGCAGT GTCAGCCCGACTCCTCAACC CTGGGCGGTG GGTCAGGAGG TGGGTCAG 180 GGTGGATCCG GAGGTGGCACCCAGGACTGC TCCTTCCAAC ACAGCCCCAT CTCCTCCG 240 TTCGCTGTCA AAATCCGTGAGCTGTCTGAC TACCTGCTTC AAGATTACCC AGTCACCG 300 GCCTCCAACC TGCAGGACGAGGAGCTCTGC GGGGGCCTCT GGCGGCTGGT CCTGGCAC 360 CGCTGGATGG AGCGGCTCAAGACTGTCGCT GGGTCCAAGA TGCAAGGCTT GCTGGAGC 420 GTGAACACGG AGATACACTTTGTCACCAAA TGTGCCTTTC AGCCC 465 465 base pairs nucleic acid singlelinear 141 GCCCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGAGCAGCTGGT 60 GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCTGCAGTGTC 120 CCCGACTCCT CAACCCTGGG CGGTGGGTCA GGAGGTGGGT CAGGAGGTGGATCCGGAG 180 GGCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGCTGTCAAAA 240 CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTCCAACCTGC 300 GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTGGATGGAGC 360 CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAACACGGAGA 420 CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTT 465 465base pairs nucleic acid single linear 142 GCCACCAACA TCTCCCGCCTCCTGCAGGAG ACCTCCGAGC AGCTGGTGGC GCTGAAGCC 60 TGGATCACTC GCCAGAACTTCTCCCGGTGC CTGGAGCTGC AGTGTCAGCC CGACTCCT 120 ACCCTGGGCG GTGGGTCAGGAGGTGGGTCA GGAGGTGGAT CCGGAGGTGG CACCCAGG 180 TGCTCCTTCC AACACAGCCCCATCTCCTCC GACTTCGCTG TCAAAATCCG TGAGCTGT 240 GACTACCTGC TTCAAGATTACCCAGTCACC GTGGCCTCCA ACCTGCAGGA CGAGGAGC 300 TGCGGGGGCC TCTGGCGGCTGGTCCTGGCA CAGCGCTGGA TGGAGCGGCT CAAGACTG 360 GCTGGGTCCA AGATGCAAGGCTTGCTGGAG CGCGTGAACA CGGAGATACA CTTTGTCA 420 AAATGTGCCT TTCAGCCCCCCCCCAGCTGT CTTCGCTTCG TCCAG 465 134 amino acids amino acid single linearNone 143 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr ProVal 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly LeuTrp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr ValAla 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu IleHis 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys LeuArg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser GluGln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe SerArg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro 130 139 amino acidsamino acid single linear None 144 Thr Gln Asp Cys Ser Phe Gln His SerPro Ile Ser Ser Asp Phe Ala 1 5 10 15 Val Lys Ile Arg Glu Leu Ser AspTyr Leu Leu Gln Asp Tyr Pro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln AspGlu Glu Leu Cys Gly Gly Leu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg TrpMet Glu Arg Leu Lys Thr Val Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu LeuGlu Arg Val Asn Thr Glu Ile His 65 70 75 80 Phe Val Thr Lys Cys Ala PheGln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser ArgLeu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110 Val Ala Leu Lys Pro TrpIle Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125 Glu Leu Gln Cys GlnPro Asp Ser Ser Thr Leu 130 135 209 amino acids amino acid single linearNone 145 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr ProVal 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly LeuTrp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr ValAla 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu IleHis 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys LeuArg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser GluGln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe SerArg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu ProPro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr AlaPro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro Val GlyLeu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln Arg ThrArg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro Val ProSer Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 402 base pairsnucleic acid single linear 146 ACCCAGGACT GCTCCTTCCA ACACAGCCCCATCTCCTCCG ACTTCGCTGT CAAAATCCG 60 GAGCTGTCTG ACTACCTGCT TCAAGATTACCCAGTCACCG TGGCCTCCAA CCTGCAGG 120 GAGGAGCTCT GCGGGGGCCT CTGGCGGCTGGTCCTGGCAC AGCGCTGGAT GGAGCGGC 180 AAGACTGTCG CTGGGTCCAA GATGCAAGGCTTGCTGGAGC GCGTGAACAC GGAGATAC 240 TTTGTCACCA AATGTGCCTT TCAGCCCCCCCCCAGCTGTC TTCGCTTCGT CCAGACCA 300 ATCTCCCGCC TCCTGCAGGA GACCTCCGAGCAGCTGGTGG CGCTGAAGCC CTGGATCA 360 CGCCAGAACT TCTCCCGGTG CCTGGAGCTGCAGTGTCAGC CC 402 630 base pairs nucleic acid single linear 147ACCCAGGACT GCTCCTTCCA ACACAGCCCC ATCTCCTCCG ACTTCGCTGT CAAAATCCG 60GAGCTGTCTG ACTACCTGCT TCAAGATTAC CCAGTCACCG TGGCCTCCAA CCTGCAGG 120GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG GTCCTGGCAC AGCGCTGGAT GGAGCGGC 180AAGACTGTCG CTGGGTCCAA GATGCAAGGC TTGCTGGAGC GCGTGAACAC GGAGATAC 240TTTGTCACCA AATGTGCCTT TCAGCCCCCC CCCAGCTGTC TTCGCTTCGT CCAGACCA 300ATCTCCCGCC TCCTGCAGGA GACCTCCGAG CAGCTGGTGG CGCTGAAGCC CTGGATCA 360CGCCAGAACT TCTCCCGGTG CCTGGAGCTG CAGTGTCAGC CCGACTCCTC AACCCTGC 420CCCCCATGGA GTCCCCGGCC CCTGGAGGCC ACAGCCCCGA CAGCCCCGCA GCCCCCTC 480CTCCTCCTAC TGCTGCTGCC CGTGGGCCTC CTGCTGCTGG CCGCTGCCTG GTGCCTGC 540TGGCAGAGGA CGCGGCGGAG GACACCCCGC CCTGGGGAGC AGGTGCCCCC CGTCCCCA 600CCCCAGGACC TGCTGCTTGT GGAGCACTGA 630 29 amino acids amino acid singlelinear 148 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly GlySer 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 20 2521 amino acids amino acid single linear 149 Pro Pro Pro Trp Ser Pro ArgPro Leu Gly Ala Thr Ala Pro Thr Ala 1 5 10 15 Gly Gln Pro Pro Leu 20 15amino acids amino acid single linear 150 Pro Pro Pro Trp Ser Pro Arg ProLeu Gly Ala Thr Ala Pro Thr 1 5 10 15 16 amino acids amino acid singlelinear 151 Val Glu Thr Val Phe His Arg Val Ser Gln Asp Gly Leu Leu ThrSer 1 5 10 15

We claim the following:
 1. A method of stimulating the production ofhematopoietic cells in a patient comprising the step of administering apolypeptide to the patient wherein the polypeptide is a human flt-3receptor agonist polypeptide comprising a modified flt-3 ligand aminoacid sequence selected from the group consisting of: (i) the sequence ofSEQ ID NO: 144; and (ii) a polypeptide comprising residues 1-132 of SEQID NO:144; wherein the modification comprises the linear rearrangementof the sequences of (i) or (ii); wherein the N-terminus is joined to theC-terminus directly or through a linker capable of joining theN-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹).
 2. A method ofstimulating the production of hematopoietic cells in a patientcomprising the step of administering a composition to the patientwherein the composition comprises a pharmaceutically acceptable carrierand a human flt-3 receptor agonist polypeptide comprising a modifiedflt-3 ligand amino acid sequence selected from the group consisting of:(i) the sequence of SEQ ID NO: 144; and (ii) a polypeptide comprisingresidues 1-132 of SEQ ID NO:144; wherein the modification comprises thelinear rearrangement of the sequences of (i) or (ii); wherein theN-terminus is joined to the C-terminus directly or through a linkercapable of joining the N-terminus to the C-terminus and new C- andN-termini are created between the amino acid residue pairs of SEQ IDNO:144 selected from the group consisting of: 28-29, 29-30, 30-31,31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43, 64-65,65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94,94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹).
 3. A method for selective ex vivo expansion of stem cellscomprising the steps of: (a) separating hematopoietic cells from othercells; (b) culturing the separated hematopoietic cells in a culturemedium comprising a human flt-3 receptor agonist polypeptide comprisinga modified flt-3 ligand amino acid sequence selected from the groupconsisting of: (i) the sequence of SEQ ID NO: 144; and (ii) apolypeptide comprising residues 1-132 of SEQ ID NO:144; wherein themodification comprises the linear rearrangement of the sequences of (i)or (ii); wherein the N-terminus is joined to the C-terminus directly orthrough a linker capable of joining the N-terminus to the C-terminus andnew C- and N-termini are created between the amino acid residue pairs ofSEQ ID NO:144 selected from the group consisting of: 28-29, 29-30,30-31, 31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43,64-65, 65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93,93-94, 94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹); and (c) harvesting the cultured cells.
 4. A method forselective ex vivo expansion of hematopoietic cells comprising the stepsof: (a) culturing the hematopoietic cells in a culture medium comprisinga composition including a pharmaceutically acceptable carrier and ahuman flt-3 receptor agonist polypeptide comprising a modified flt-3ligand amino acid sequence selected from the group consisting of: (i)the sequence of SEQ ID NO: 144; and (ii) a polypeptide comprising theresidues 1-132 of SEQ ID NO:144; wherein the modification comprises thelinear rearrangement of the sequences of (i) or (ii); wherein theN-terminus is joined to the C-terminus directly or through a linkercapable of joining the N-terminus to the C-terminus and new C- andN-termini are created between the amino acid residue pairs of SEQ IDNO:144 selected from the group consisting of: 28-29, 29-30, 30-31,31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43, 64-65,65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94,94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹); and (b) harvesting the cultured cells.
 5. A method forselective ex vivo expansion of hematopoietic cells comprising the stepsof: (a) separating hematopoietic cells from other cells; (b) culturingthe separated hematopoietic cells in a culture medium comprising acomposition including a pharmaceutically acceptable carrier and a humanflt-3 receptor agonist polypeptide comprising a modified flt-3 ligandamino acid sequence selected from the group consisting of: (i) thesequence of SEQ ID NO: 144; and (ii) a polypeptide comprising residues1-132 of SEQ ID NO:144; wherein the modification comprises the linearrearrangement of the sequences of (i) or (ii); wherein the N-terminus isjoined to the C-terminus directly or through a linker capable of joiningthe N-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹); and (c)harvesting the cultured cells.
 6. A method for treatment of a patienthaving a hematopoietic disorder comprising the steps of: (a) removinghematopoietic cells from the patient; (b) culturing the separatedhematopoietic cells in a culture medium comprising a human flt-3receptor agonist polypeptide comprising a modified flt-3 ligand aminoacid sequence selected from the group consisting of: (i) the sequence ofSEQ ID NO: 144; and (ii) a polypeptide comprising residues 1-132 of SEQID NO:144; wherein the modification comprises the linear rearrangementof the sequences of (i) or (ii); wherein the N-terminus is joined to theC-terminus directly or through a linker capable of joining theN-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹); (c) harvestingthe cultured cells; and (d) transplanting the cultured cells into thepatient.
 7. A method for treatment of a patient having a hematopoieticdisorder comprising the steps of: (a) removing hematopoietic cells fromthe patient; (b) separating the hematopoietic cells from other cells;(c) culturing the separated hematopoietic cells in a culture mediumcomprising a human flt-3 receptor agonist polypeptide comprising amodified flt-3 ligand amino acid sequence selected from the groupconsisting of: (i) the sequence of SEQ ID NO: 144; and (ii) apolypeptide comprising residues 1-132 of SEQ ID NO:144; wherein themodification comprises the linear rearrangement of the sequences of (i)or (ii); wherein the N-terminus is joined to the C-terminus directly orthrough a linker capable of joining the N-terminus to the C-terminus andnew C- and N-termini are created between the amino acid residue pairs ofSEQ ID NO:144 selected from the group consisting of: 28-29, 29-30,30-31, 31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43,64-65, 65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93,93-94, 94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹); (d) harvesting the cultured cells; and (e) transplanting thecultured cells into the patient.
 8. A method for treatment of a patienthaving a hematopoietic disorder, comprising the steps of: (a) removinghematopoietic cells from the patient; (b) culturing the hematopoieticcells in a growth medium comprising a human flt-3 receptor agonistpolypeptide comprising a modified flt-3 ligand amino acid sequenceselected from the group consisting of: (i) the sequence of SEQ ID NO:144; and (ii) a polypeptide comprising residues 1-132 of SEQ ID NO:144;wherein the modification comprises the linear rearrangement of thesequences of (i) or (ii); wherein the N-terminus is joined to theC-terminus directly or through a linker capable of joining theN-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹); (c) harvestingthe cultured cells; and (d) transplanting the cultured cells into thepatient.
 9. A method for treatment of a patient having a hematopoieticdisorder, comprising the steps of: (a) removing hematopoietic cells fromthe patient; (b) separating hematopoietic cells from other cells; (c)culturing the separated hematopoietic cells in a growth mediumcomprising a composition including a pharmaceutically acceptable carrierand a human flt-3 receptor agonist polypeptide comprising a modifiedflt-3 ligand amino acid sequence selected from the group consisting of:(i) the sequence of SEQ ID NO: 144; and (ii) a polypeptide comprisingresidues 1-132 of SEQ ID NO:144; wherein the modification comprises thelinear rearrangement of the sequences of (i) or (ii); wherein theN-terminus is joined to the C-terminus directly or through a linkercapable of joining the N-terminus to the C-terminus and new C- andN-termini are created between the amino acid residue pairs of SEQ IDNO:144 selected from the group consisting of: 28-29, 29-30, 30-31,31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43, 64-65,65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94,94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹); (d) harvesting the cultured cells; and (e) transplanting thecultured cells into the patient.
 10. A method of human gene therapycomprising the steps of: (a) removing hematopoietic cells from apatient; (b) culturing the hematopoietic cells in a growth mediumcomprising a human flt-3 receptor agonist polypeptide comprising amodified flt-3 ligand amino acid sequence selected from the groupconsisting of: (i) the sequence of SEQ ID NO: 144; and (ii) apolypeptide comprising residues 1-132 of SEQ ID NO:144; wherein themodification comprises the linear rearrangement of the sequences of (i)or (ii); wherein the N-terminus is joined to the C-terminus directly orthrough a linker capable of joining the N-terminus to the C-terminus andnew C- and N-termini are created between the amino acid residue pairs ofSEQ ID NO:144 selected from the group consisting of: 28-29, 29-30,30-31, 31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43,64-65, 65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93,93-94, 94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹); (c) transducing the cultured cells with DNA; (d) harvestingthe transduced cells; and (e) transplanting the transduced cells intothe patient.
 11. A method of human gene therapy comprising the steps of:(a) removing hematopoietic cells from a patient; (b) separating thehematopoietic cells from other cells; (c) culturing the separatedhematopoietic cells in a growth medium comprising a human flt-3 receptoragonist polypeptide comprising a modified flt-3 ligand amino acidsequence selected from the group consisting of: (i) the sequence of SEQID NO: 144; and (ii) a polypeptide comprising residues 1-132 of SEQ IDNO:144; wherein the modification comprises the linear rearrangement ofthe sequences of (i) or (ii); wherein the N-terminus is joined to theC-terminus directly or through a linker capable of joining theN-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹) ; (d)transducing the cultured cells with DNA; (e) harvesting the transducedcells; and (f) transplanting the transduced cells into the patient. 12.A method of human gene therapy comprising the steps of: (a) removinghematopoietic cells from a patient; (b) separating the hematopoieticcells from other cells; (c) culturing the separated hematopoietic cellsin a growth medium comprising a composition including a pharmaceuticallyacceptable carrier and a human flt-3 receptor agonist polypeptidecomprising a modified flt-3 ligand amino acid sequence selected from thegroup consisting of: (i) the sequence of SEQ ID NO: 144; and (ii) apolypeptide comprising residues 1-132 of SEQ ID NO:144; wherein themodification comprises the linear rearrangement of the sequences of (i)or (ii); wherein the N-terminus is joined to the C-terminus directly orthrough a linker capable of joining the N-terminus to the C-terminus andnew C- and N-termini are created between the amino acid residue pairs ofSEQ ID NO:144 selected from the group consisting of: 28-29, 29-30,30-31, 31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43,64-65, 65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93,93-94, 94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹); (d) transducing the cultured cells with DNA; (e) harvestingthe transduced cells; and (f) transplanting the transduced cells intothe patient.
 13. A method of human gene therapy comprising the steps of:(a) removing hematopoietic cells from a patient; (b) separating thehematopoietic cells from other cells; (c) culturing the separatedhematopoietic cells in a growth medium comprising a compositionincluding a pharmaceutically acceptable carrier and a human flt-3receptor agonist polypeptide comprising a modified flt-3 ligand aminoacid sequence selected from the group consisting of: (i) the sequence ofSEQ ID NO: 144; and (ii) a polypeptide comprising residues 1-132 of SEQID NO:144; wherein the modification comprises the linear rearrangementof the sequences of (i) or (ii); wherein the N-terminus is joined to theC-terminus directly or through a linker capable of joining theN-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹) (d) transducingthe cultured cells with DNA; (e) harvesting the transduced cells; and(f) transplanting the transduced cells into the patient.
 14. A methodfor the production of dendritic cells comprising the steps of: (a)separating hematopoietic progenitor cells or CD34+ cells from othercells; and (b) culturing the hematopoietic progenitor cells or CD34+cells in a growth medium comprising a human flt-3 receptor agonistpolypeptide comprising a modified flt-3 ligand amino acid sequenceselected from the group consisting of: (i) the sequence of SEQ ID NO:144; and (ii) a polypeptide comprising residues 1-132 of SEQ ID NO:144;wherein the modification comprises the linear rearrangement of thesequences of (i) or (ii); wherein the N-terminus is joined to theC-terminus directly or through a linker capable of joining theN-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹).
 15. The methodof claim 14 further comprising the step of pulsing the culturinghematopoietic progenitor cells or CD34+cells with an antigen.
 16. Themethod of claim 14 wherein the growth medium further comprises one ormore factors selected from the group consisting of: GM-CSF, IL-4, TNF-α,stem cell factor (SCF), flt-3 ligand, IL-3, an IL-3 variant, an IL-3variant fusion protein, and a multi-functional receptor agonist.
 17. Themethod of claim 15 wherein the growth medium further comprises one ormore factors selected from the group consisting of: GM-CSF, IL-4, TNF-α,stem cell factor (SCF), flt-3 ligand, IL-3, an IL-3 variant, an IL-3variant fusion protein, and a multi-functional receptor agonist.
 18. Amethod for treating a human having a tumor, infection or auto-immunedisease comprising the step of administering a human flt-3 receptoragonist polypeptide comprising a modified flt-3 ligand amino acidsequence selected from the group consisting of: (i) the sequence of SEQID NO: 144; and (ii) a polypeptide comprising residues 1-132 of SEQ IDNO:144; wherein the modification comprises the linear rearrangement ofthe sequences of (i) or (ii); wherein the N-terminus is joined to theC-terminus directly or through a linker capable of joining theN-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹) to the human.19. The method of claim 18 further comprising administrating one or morefactors selected from the group consisting of: GM-CSF, IL-4, TNF-α, stemcell factor (SCF), flt-3 ligand, IL-3, an IL-3 variant, an IL-3 variantfusion protein, and a multi-functional receptor agonist.
 20. The methodof claim 18 further comprising the step of administering an antigen tothe patient.
 21. The method of claim 19 further comprising the step ofadministering an antigen to the patient.
 22. A method for treating ahuman having a tumor, infection or auto-immune disease, comprising thesteps of: (a) mobilizing dendritic cell progenitors or mature dendriticcells by administering a human flt-3 receptor agonist polypeptidecomprising a modified flt-3 ligand amino acid sequence selected from thegroup consisting of: (i) the sequence of SEQ ID NO: 144; and (ii) apolypeptide comprising residues 1-132 of SEQ ID NO:144; wherein themodification comprises the linear rearrangement of the sequences of (i)or (ii); wherein the N-terminus is joined to the C-terminus directly orthrough a linker capable of joining the N-terminus to the C-terminus andnew C- and N-termini are created between the amino acid residue pairs ofSEQ ID NO:144 selected from the group consisting of: 28-29, 29-30,30-31, 31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43,64-65, 65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93,93-94, 94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹) to the human; (b) removing the dendritic cell precursors ormature dendritic cells by a blood draw or pheresis; (c) pulsing thedendritic cell precursors or mature dendritic cells with an antigen; and(d) returning the antigen pulsed dendritic cell precursors or maturedendritic cells to the human.
 23. The method of claim 22 furthercomprising administering in step (a) one or more factors selected fromthe group consisting of: GM-CSF, IL-4, TNF-α, stem cell factor (SCF),flt-3 ligand, IL-3, an IL-3 variant, an IL-3 variant fusion protein, anda multi-functional receptor agonist.
 24. The method of claim 22 furthercomprising the step of culturing said dendritic cell precursors ormature dendritic cells from step (b) in a growth medium comprising thehuman flt-3 receptor agonist polypeptide.
 25. The method of claim 23further comprising the step of culturing the dendritic cell precursorsor mature dendritic cells from step (b) in a growth medium comprisingthe human flt-3 receptor agonist polypeptide.
 26. The method of claim 24wherein the growth medium further comprises one or more factors selectedfrom the group consisting of: GM-CSF, IL-4, TNF-α, stem cell factor(SCF), flt-3 ligand, IL-3, an IL-3 variant, an IL-3 variant fusionprotein, and a multi-functional receptor agonist.
 27. The method ofclaim 25 wherein the growth medium further comprises one or more factorsselected from the group consisting of: GM-CSF, IL-4, TNF-α, stem cellfactor (SCF), flt-3 ligand, IL-3, an IL-3 variant, an IL-3 variantfusion protein, and a multi-functional receptor agonist.
 28. A methodfor treating a human having a tumor, infection or auto-immune diseasecomprising the steps of: (a) removing hematopoietic progenitor cells orCD34+ cells from the human by a blood draw or pheresis; (b) culturingthe hematopoietic progenitor cells or CD34+cells in a growth mediumcomprising a human flt-3 receptor agonist polypeptide comprising amodified flt-3 ligand amino acid sequence selected from the groupconsisting of: (i) the sequence of SEQ ID NO: 144; and (ii) apolypeptide comprising residues 1-132 of SEQ ID NO:144; wherein themodification comprises the linear rearrangement of the sequences of (i)or (ii); wherein the N-terminus is joined to the C-terminus directly orthrough a linker capable of joining the N-terminus to the C-terminus andnew C- and N-termini are created between the amino acid residue pairs ofSEQ ID NO:144 selected from the group consisting of: 28-29, 29-30,30-31, 31-32, 32-33, 34-35, 36-37, 37-38, 38-39, 40-41, 41-42, 42-43,64-65, 65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93,93-94, 94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, and102-103; and wherein optionally the flt-3 receptor agonist polypeptideis immediately preceded by (methionine⁻¹), (alanine⁻¹) or (methionine⁻²,alanine⁻¹) to produce dendritic cell precursors or mature dendriticcells; and (c) returning the dendritic cell precursors or maturedendritic cells to the human.
 29. A method for treating a human having atumor, infection or auto-immune disease comprising the steps of: (a)removing hematopoietic progenitor cells or CD34+ cells from the patientby a blood draw or pheresis; (b) culturing the hematopoietic progenitorcells or CD34+ cells in a growth medium comprising a human flt-3receptor agonist polypeptide comprising a modified flt-3 ligand aminoacid sequence selected from the group consisting of: (i) the sequence ofSEQ ID NO: 144; and (ii) a polypeptide comprising residues 1-132 of SEQID NO:144; wherein the modification comprises the linear rearrangementof the sequences of (i) or (ii); wherein the N-terminus is joined to theC-terminus directly or through a linker capable of joining theN-terminus to the C-terminus and new C- and N-termini are createdbetween the amino acid residue pairs of SEQ ID NO:144 selected from thegroup consisting of: 28-29, 29-30, 30-31, 31-32, 32-33, 34-35, 36-37,37-38, 38-39, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88,88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98,98-99, 99-100, 100-101, 101-102, and 102-103; and wherein optionally theflt-3 receptor agonist polypeptide is immediately preceded by(methionine⁻¹), (alanine⁻¹) or (methionine⁻², alanine⁻¹) to producedendritic cell precursors or mature dendritic cells; (c) pulsing thedendritic cell precursors or mature dendritic cells with an antigen; and(d) returning the antigen pulsed dendritic cell precursors or maturedendritic cells to the human.
 30. The method of claim 28 furthercomprising the step of separating the hematopoietic progenitor cells orCD34+ cells from other cells prior to culturing.
 31. The method of claim29 further comprising the step of separating the hematopoieticprogenitor cells or CD34+ cells from other cells prior to culturing. 32.The method of claim 28 wherein the culture medium further comprises oneor more factors selected from the group consisting of: GM-CSF, IL-4,TNF-α, stem cell factor (SCF), flt-3 ligand, IL-3, an IL-3 variant, anIL-3 variant fusion protein, and a multi-functional receptor agonist.33. The method of claim 29 wherein the culture medium further comprisesone or more factors selected from the group consisting of: GM-CSF, IL-4,TNF-α, stem cell factor (SCF), flt-3 ligand, IL-3, an IL-3 variant, anIL-3 variant fusion protein, and a multi-functional receptor agonist.34. The method of claim 30 wherein the culture medium further comprisesone or more factors selected from the group consisting of: GM-CSF, IL-4,TNF-α, stem cell factor (SCF), flt-3 ligand, IL-3, an IL-3 variant, anIL-3 variant fusion protein, and a multi-functional receptor agonist.35. The method of claim 31 wherein the culture medium further comprisesone or more factors selected from the group consisting of: GM-CSF, IL-4,TNF-α, stem cell factor (SCF), flt-3 ligand, IL-3, an IL-3 variant, anIL-3 variant fusion protein, and a multi-functional receptor agonist.